Decreased Nrf2 protein level and low sperm quality in intractable spermatocystitis

Abstract

To investigate the molecular etiology of low sperm quality in patients with intractable spermatocystitis, spermatozoa samples from patients with persistent hematospermia undergoing transurethral seminal vesiculoscopy and healthy volunteers were utilized. Spermatozoa samples were collected from the seminal vesicles through transurethral seminal vesiculoscopy or by masturbation ejaculation. Sperm quality was analyzed by a WLJY-9000 color semen analysis system. Measurement of tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) in the seminal plasma was performed using enzyme-linked immunosorbent assay (ELISA). Measurement of H₂O₂ in the seminal plasma was performed with a hydrogen peroxide kit. The protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and phosphorylated-Nrf2 (p-Nrf2) were measured by western blot analysis and immunofluorescence assays. Low sperm quality parameters and increased levels of inflammatory cytokines (TNFα, IL-6, and H₂O₂) in the seminal plasma were detected among the semen samples from the patients with persistent hematospermia. Nrf2 and p-Nrf2 were strongly expressed in the nucleus and periphery of human sperm cells, according to the results of the immunofluorescence assays. The protein levels of Nrf2 and p-Nrf2 were significantly lower in the spermatozoa samples from patients with persistent hematospermia than in those from healthy volunteers with normal sperm motility. The results suggested that Nrf2 signaling might play a role in the low sperm quality of patients with intractable spermatocystitis.

INTRODUCTION

Seminal vesiculitis, accompanied by hematospermia, is a common inflammatory disease of the seminal vesicle. Most cases are caused by bacterial infections, though they can have nonbacterial reasons.1 The seminal vesicle gland, the prostate gland, the bulbar urethral gland, and the paraurethral gland constitute the accessory glands, which are the internal reproductive organs in men. The seminal vesicle gland is an elliptical muscular sac located above the prostate, on the lateral side of the ampulla of the vas deferens, between the base of the bladder and the rectum. The terminal excretory tube of the seminal vesicle and the vas deferens join to form the ejaculatory tube, which opens into the urethra in the prostatic part of the urethra. Seminal vesiculitis can result in increased reactive oxygen species (ROS) production in the seminal plasma, and the ROS in the seminal plasma may cause sperm membrane damage, leading to low sperm motility.2 Castiglione et al.2 showed that infertile patients with bacterial bilateral prostato-vesiculitis have lower sperm quality than men with bacterial prostatitis alone or healthy fertile men. ROS are increased in infertile men with prostato-vesiculitis compared to infertile men with bacterial prostatitis or healthy fertile men. The increased ROS could cause more sperm membrane damage. Previous studies have shown that ROS play an important role in male infertility and that excessive ROS levels are negatively correlated with the quality of spermatozoa and their overall fertilization ability.3 Many studies have indicated that the oxidative stress reaction caused by infection or inflammation decreases sperm motility.4,5 Zhu et al.6 demonstrated that Toll-like receptor (TLR) activation in sperm reduces sperm motility by signaling through myeloid differentiation factor 88, phosphatidylinositol 3-kinase, and glycogen synthase kinase-3α. Hagan et al.7 found that the protein levels of TLR-2, TLR-4, cyclooxygenase-2 (COX-2), and nuclear factor erythroid 2-related factor 2 (Nrf2) are significantly higher in the semen of leukocytospermia patients than in that of men without this condition.7 Currently, a large number of biomarkers for spermatogenesis, such as transient receptor potential vanilloid type 1 (TRPV1), N6-methyladenosine, protamine 2, and caspase 9, have been reported.8,9,10 Nrf2 is a cotranscription factor that is normally tagged for degradation in the cytosol by Kelch-like ECH-associated protein 1 (Keap1). Once Nrf2 is activated, it pairs with a Maf protein, and the resulting heterodimer binds to the antioxidant response elements (AREs) in the upstream promoter region of antioxidant genes such as glutathione-S-transferase (GST), heme oxygenase 1 (HMOX1), and thioredoxin reductase 1 (TXNRD1), initiating their transcription. Phosphorylated-Nrf2 (p-Nrf2) is the Nrf2 protein phosphorylated at a site in the Neh2 domain. Kinases such as protein kinase C (PKC) can phosphorylate Nrf2 in the Neh2 domain to release Nrf2 from Keap1, promoting Nrf2 transcription activity.11 Elevated p-Nrf2 levels suggest that the Nrf2 pathway is activated.11 Chen et al.12 found that the levels of Nrf2 mRNA were low in spermatozoa from men with low sperm motility. In addition to its regulation by Keap1, Nrf2 can also respond to ROS attack by activating the transcription of antioxidant enzymes by binding directly to AREs.13 The specific mechanisms by which Nrf2 affects sperm motility and infertility remain unclear. We aimed to identify whether the physiopathological mechanism of low sperm quality and infertility in patients with nonbacterial seminal vesiculitis was associated with the Nrf2 protein in sperm.

PARTICIPANTS AND METHODS

Study population and sperm sample collection

The present study was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (Wuhan, China; Approval No. 2017110). From 01 January 2018 to 30 December 2019, there were 32 healthy volunteers and 35 patients with seminal vesiculitis initially included in the study from our outpatients and inpatients in Wuhan and surrounding regions. Two healthy control volunteers and five patients were excluded due to the failure of semen collection. An informed consent form was signed by all participants. The spermatozoa samples were collected from patients with seminal vesiculitis who were diagnosed by magnetic resonance and transurethral seminal vesiculoscopy. The collection of abnormal spermatozoa samples was conducted during transurethral seminal vesiculoscopy (Richard Wolf, Knittlingen, Germany) with a rod body length of 315 mm and channel diameter of 3.3 Fr. When the endoscopic rod was inserted into the internal seminal vesicle, seminal vesicle fluid was collected with a 5 ml injection syringe through the 3.3 Fr vesiculoscopy drainage channel. The flushing flow was stopped before the fluid sample collection. Control sperm samples with normal sperm quality parameters were obtained from healthy volunteers through masturbation ejaculation after abstinence for 5 days. Highly purified spermatozoa were obtained using discontinuous Percoll density gradient centrifugation of the semen samples collected through the above two methods. Approximately 1 ml of semen was layered on top of equal volumes of 40% Percoll solution (upper layer) and 80% Percoll solution (lower layer), which formed discontinuous density gradients, in a 15 ml centrifuge tube and centrifuged (TD6M, Xinlin, Zhengzhou, China) at 300g for 20 min. Subsequently, the sperm separated from other semen components through centrifugation were collected. The purified sperm were characterized based on the detection of mRNA biomarkers, including TLRs and protamine-2 (PRM2).

RNA assay for sperm identification

Reverse transcription–polymerase chain reaction (RT-PCR) was performed for sperm identification. Portions of purified spermatozoa were dissolved in the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) to extract total RNA according to the manufacturer’s instructions. A ReverTra Ace qPCR RT kit (Toyobo, Tokyo, Japan) was used to synthesize the cDNA. The reaction contained 4 μl of 5× buffer, 1 μl of reverse transcriptase, 1 μg of total RNA, and RNase-free H₂O to a total volume of 20 μl. The relative expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PRM2, TLR2, and TLR4 were determined by RT-PCR using an RT-PCR kit (Thermo Fisher Scientific, Shanghai, China). The reactions contained 10 μl of Taq PCR mix, 1 μl of cDNA, 1 μl of primer (50 nmol l⁻¹), and 7 μl of RNase-free H₂O. The specific primer pairs are shown in Supplementary Table 1.

Supplementary Table 1.

Primers used in the reverse transcription-polymerase chain reaction for sperm identification

Name	Primer	Sequence
GAPDH	Forward	5’- AACGACCCCTTCATTGACCT-3’
	Reverse	5’- CCCCATTTGATGTTAGCGGG-3’
PRM2	Forward	5’- GGATCCACAGGCGGCAGCATCGCT-3’
	Reverse	5’- GCATGTTCTCTTCCTGGTTCTGCA-3’
TLR2	Forward	5’- GATGCCTACTGGGTGGAGAA-3’
	Reverse	5’- GAATGAGAATGGCAGCATCA-3’
TLR4	Forward	5’- CCATAAAAGCCGAAAGGTGA-3’
	Reverse	5’- CAGGGCTTTTCTGAGTCGTC-3’

GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PRM2: protamine-2; TLR2: toll-like receptor 2; TLR4: toll-like receptor 4

Semen analysis

The sperm quality was analyzed with a WLJY-9000 color semen analysis system (Beijing Wei-Li New Century Technology Development Ltd., Beijing, China).14,15 The parameters used to evaluate sperm quality, including sperm concentration, total number of spermatozoa, number of forward motility, motionless sperm, and nonforward motile sperm, were recorded. All sperm samples were placed in EP tubes to liquefy for at least 30 min before Percoll density gradient centrifugation (TD6M, Xinlin) and then sent for quality analysis.

Semen cytokine measurements

The supernatants of the semen samples were collected and stored at −80°C. TNFα and IL-6 were measured using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (Sangon Biotech, Shanghai, China).16 All ELISAs for the determination of cytokines were performed according to the manufacturer’s instructions, and the ELISAs for every sample were repeated three times. We set the minimum detectable dose threshold (MDD) for TNFα ELISA at 0.112 pg ml⁻¹ and the intra- and inter-assay coefficients of variation at <5%. The MDD was determined by adding standard deviations to the mean optical density value of 20 zero-standard replicates and calculating the corresponding concentration. The MDD in the IL-6 ELISAs was 0.035 pg ml⁻¹, and the intra- and inter-assay coefficients of variation were <5%. H₂O₂ levels in semen samples were measured using a hydrogen peroxide kit (Beyotime, Shanghai, China). Absorbance indicating the H₂O₂ level was measured at a wavelength of 240 nm in ELISA.17

Immunofluorescence assay

The sperm samples purified using the approaches described above were diluted in phosphate-buffered saline (PBS) and smeared on slides. The sperm cells on the slide were fixed in 0.025% paraformaldehyde for 20 min and then washed with PBS three times. Subsequently, the cell samples were fixed again for 5 min in 1.25% paraformaldehyde and then permeabilized with 0.5% Triton X-100 in PBS for 15 min (above reagents were bought from Zhongyi Biotechnology Co., Wuhan, China). After the slides were washed with PBS three times, they were blocked with 5% bovine serum albumin (BSA) for 20 min, and the primary antibody was incubated with the sperm in a humid chamber overnight at 4°C. The primary antibodies applied in the study were anti-Nrf2 antibody (1:100; Abcam, Boston, MA, USA) and anti-p-Nrf2 antibody (1:100; Abcam). Following incubation with the primary antibody, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG second antibody (Beyotime) was incubated with the sperm on the slides. 1,4-diazabicyclo[2.2.2]octane (DABCO; Sigma-Aldrich, St. Louis, MI, USA) was applied to protect cell fluorescence from quenching after 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) was used to stain the nucleus of sperm. Finally, the slides were covered with a coverslip and observed by confocal microscopy (Nikon, Tokyo, Japan). For each sample, five microscope fields, including sperm were randomly acquired by the camera. The fluorescence intensity of the pictures was analyzed by using Image-Pro-Plus (IPP) software (Media Cybernetics, Baltimore, MD, USA).7

Western blot

The purified sperm cells obtained by Percoll gradient centrifugation were lysed in triple-detergent radioimmunoprecipitation assay (RIPA) buffer consisting of 50 mmol l⁻¹ Tris (pH 7.4), 150 mmol l⁻¹ NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfonate (SDS), and general protease and phosphatase inhibitors (Above reagents were bought from Zhongyi Biotechnology Co.). One milliliter of RIPA lysis buffer was used for 0.5 × 10⁶–5 × 10⁶ cells. After lysis, the suspension was centrifuged at 14 000g for 5 min at 4°C, and the protein supernatant was transferred to a new tube and saved at −80°C for further analysis. Cytoplasmic and nuclear proteins in collected sperm were extracted with a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Protein quantity was determined by the bicinchoninic acid (BCA) method (Beyotime). Equal quantities of protein (40 µg) were denatured at 95°C for 5 min, electrophoresis was performed on a 12% discontinuous SDS–polyacrylamide gel, and then the protein was transferred to a PVDF membrane (Sigma-Aldrich). The membranes were reacted with blocking buffer (5% skim milk in Tris-buffered saline containing 0.5% [v/v] Tween 20 [TBST] buffer) for 30 min at ambient temperature and incubated with anti-Nrf2 antibody (1:1000; Abcam) and anti-p-Nrf2 antibody (1:10 000; Abcam) overnight. H3 antibody (1:1000; Abcam) and GAPDH antibody (1:1000; Bioworld, Nanjing, China) were used to detect reference nuclear and total protein levels, respectively. Following incubation with secondary antibodies at ambient temperature for 1 h, signals were visualized by enhanced chemiluminescence.7 The optical density (OD) values of the Nrf2 and p-Nrf2 protein bands were analyzed relative to the OD value of the GAPDH band under the same condition.

Statistical analyses

The Student’s t-test method was used for the statistical analysis of continuous data with equal variance between the seminal vesiculitis group and the healthy volunteer group. Analysis of variance was applied for the statistical analysis of continuous data with unequal variance between the seminal vesiculitis group and the healthy volunteer group. P < 0.05 indicated statistical significance.

RESULTS

Participant characteristics

We reviewed the cases in our hospital to select the patients diagnosed with seminal vesiculitis with persistent hematospermia. The included cases were confirmed by magnetic resonance and transurethral seminal vesiculoscopy. Five patients were excluded because semen could not be collected during seminal vesiculoscopy. Healthy volunteers with normal results on routine semen analysis in the andrology clinic were invited to donate their semen for further research. Thirty-two healthy volunteers were approached initially, and 2 were excluded due to semen collection failure. A total of 60 sperm samples from 60 males (30 patients with seminal vesiculitis and 30 healthy volunteers) were utilized in our study for analysis of the expression levels of Nrf2 and p-Nrf2 in patients with persistent hematospermia and those with normal sperm quality. Sperm from the seminal vesiculitis group with persistent hematospermia were identified as having significantly lower sperm quality than those from the control group. The specific characteristics of these participants are presented in Table 1.

Table 1.

The characteristics in the quality of spermatozoa of the included patients

Variable	Control group (n=30)	Seminal vesiculitis group (n=30)	P
Age (year), mean (s.d.)	30.4 (4.7)	31.7 (4.8)	0.296
Days of abstinence	5	5	-
Sample	Semen of healthy volunteers (ejaculate)	Semen from SV patients collected via TSV	-
Sperm concentration (×106 ml−1), mean (s.d.)	39.43 (23.22)	15.74 (29.31)	0.001
Total number of spermatozoa (×106), mean (s.d.)	205.95 (190.32)	85.64 (182.53)	0.015
Forward motility (%), mean (s.d.)	63.3 (60.9)	7.9 (66.8)	0.001
Normal forms (%), mean (s.d.)	8.2 (9.2)	3.2 (8.6)	0.032
IM (%), mean (s.d.)	28.5 (98.5)	88.9 (100.3)	0.021
PR + NP (%), mean (s.d.)	71.5 (71.3)	11.1 (74.4)	0.002

IM: motionless sperm; PR: progressive motility; NP: nonprogressive motility; SV: seminal vesiculitis; TSV: transurethral seminal vesiculoscopy; s.d.: standard deviation; -: no value

Separation and identification of sperm

Sperm separated from the seminal fluid using discontinuous Percoll density gradient centrifugation was observed under a light microscope. The RT-PCR results revealed that PRM2 mRNA was remarkably expressed in the sperm. The mRNA TLR2 and TLR4 were also detected in the total RNA extraction (Figure 1).

Figure 1.

Purified sperm was separated from seminal fluid using discontinuous Percoll density gradient centrifugation and RT-PCR showed that PRM2, TLR2, and TLR4 mRNA were obviously expressed in the separated spermatozoa. PRM2: protamine-2; TLR2: toll-like receptor 2; TLR4: toll-like receptor 4; RT-PCR: reverse transcription-polymerase chain reaction.

The levels of TNFα, IL-6, and H₂O₂ in seminal plasma

The levels of TNFα and IL-6 in the seminal plasma were significantly elevated in the semen samples of patients with persistent hematospermia compared to the semen samples of normal healthy males (both P < 0.001; Figure 2a and 2b). H₂O₂ levels were significantly increased in the seminal plasma of patients with persistent hematospermia (P < 0.05; Figure 2c).

Figure 2.

The levels of TNFα, IL-6, and H₂O₂ detected in the seminal plasma collected from healthy males and patients with spermatocystitis suffering from persistent hematospermia. (a) IL-6 and (b) TNFα were significantly elevated in the semen samples of patients with spermatocystitis compared to the semen samples of normal healthy males. (c) H₂O₂ levels were significantly increased in the seminal plasma of patients with spermatocystitis. ^*P < 0.05; ^***P < 0.001. TNFα: tumor necrosis factor alpha; IL-6: interleukin-6; SV: seminal vesiculitis.

The protein levels of Nrf2 and p-Nrf2 in sperm

Immunofluorescence assays indicated that the protein levels of Nrf2 and p-Nrf2 in the sperm were significantly decreased in the group with low sperm quality compared to the control group with normal sperm motility (Figure 3 and 4). Nrf2 and p-Nrf2 were located in the sperm cytoplasm and nuclei (more notably in the sperm nuclei; Figure 3–5). Western blot assays also showed significantly decreased levels of Nrf2 and p-Nrf2 in the group with low sperm quality compared to the control group with normal sperm motility (Figure 3 and 4).

Figure 3.

(a) Immunofluorescence assay for the detection of Nrf2 protein levels of sperm in the SV group and the control group (30 × 5 = 150 images analyzed in each group). (b) Statistical analysis of the immunofluorescence assay suggested that the Nrf2 protein levels in sperm were significantly decreased in the SV group compared to the control group. (c) Statistical analysis and (d) representative bands for western blot assay show that Nrf2 protein levels were significantly decreased in the SV group compared to the control group. SV: seminal vesiculitis; Nrf2: nuclear factor erythroid 2-related factor 2; DAPI: 4’,6-diamidino-2-phenylindole. ^*P < 0.05.

Figure 4.

(a) Immunofluorescence assay for the detection of p-Nrf2 protein levels in sperm in the SV group and the control group (30 × 5 = 150 images analyzed in each group). (b) Statistical analysis of the immunofluorescence assay suggested that p-Nrf2 protein levels in sperm were significantly decreased in the SV group compared to the control group. (c) Statistical analysis and (d) representative bands for western blot assay showed that p-Nrf2 protein levels were significantly decreased in the SV group compared to the control group. SV: seminal vesiculitis; Nrf2: nuclear factor erythroid 2-related factor 2; p-Nrf2: phosphorylated-Nrf2; DAPI: 4’,6-diamidino-2-phenylindole. ^*P < 0.05.

Figure 5.

Nrf2 protein localized to the cytoplasm and cell nucleus in sperm (more notably in the nuclei). (a) Immunofluorescence image of FITC-labeled Nrf2 in sperm (white arrow indicates the expression and location of Nrf2 protein). (b) Western blot shows that Nrf2 was expressed in the sperm cytoplasm and nucleus. Nrf2: nuclear factor erythroid 2-related factor 2; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

DISCUSSION

Our study found that both Nrf2 and p-Nrf2 protein levels were significantly decreased in sperm samples from patients with persistent hematospermia compared to sperm samples from normal healthy males. In addition, the levels of TNFα and IL-6 in the seminal plasma were significantly elevated in the experimental group (patients with persistent hematospermia) compared to the control group. The Nrf2 signaling pathway, as an important antioxidative signaling pathway, might play an important role in the pathological process responsible for the lower semen quality in patients with persistent hematospermia.

Low sperm quality is typical in patients with intractable spermatocystitis. In these patients, the progression of sperm through the sperm duct is commonly completely obstructed or incompletely blocked. Long-term inflammation in the seminal vesicle creates persistent hematospermia, and the inflammation itself is also harmful to sperm quality, even leading to infertility.18,19 The pathological process of infection inflammation affects sperm function through various mechanisms. On the one hand, pro-inflammatory cytokines such as IL-1, IL-2, and IL-6 produced by locally activated cells regulate the physiological levels of ROS. Elevated levels of ROS caused by inflammatory mediators have a toxic effect on human spermatozoa, which results in damage to mtDNA and decreased sperm mitochondria numbers.20 On the other hand, bacterial inflammation has a harmful effect on sperm function through the TLR signaling pathway.21 Zhu et al.6 reported that lipopolysaccharide (LPS) is able to bind to TLRs, leading to the activation of the TLR signaling pathway, which affects sperm mitochondrial function and motility through phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase-3α (GSK-3α).

There may be a defense system in the human body that can increase the levels of cytoprotective enzyme-encoding genes once the body suffers some harmful stimulation.22 Nrf2 plays a central role in the regulation of the inducible expression of cellular defense enzymes. As a member of the cap-n-collar (CNC) subfamily of basic region-leucine zipper-type transcription factors (bZIP-TFs), Nrf2 is constantly ubiquitinated in the cytoplasm by a cellular stress sensor protein, Keap1, after which it is degraded by the proteasome. Oxidative and electrophilic reagents covalently modify the specific Cys residues of Keap1, which leads to the release and stabilization of Nrf2. Stabilized Nrf2 then translocates to the nucleus and forms heterodimers with small Maf proteins (sMaf, another group of bZIP-TFs).23 Nrf2 functions through the binding of its Nrf2-sMaf heterodimer to AREs located in the regulatory regions of many cytoprotective enzyme-encoding genes. Several hundred Nrf2 target genes are involved in detoxification, antioxidation, and metabolism. Nrf2 is known to attenuate inflammation and antioxidation function.24 Thus, it can be hypothesized that Nrf2, as a cotranscription factor that regulates antioxidant enzyme levels, may protect sperm from the toxic effects of ROS and inflammation. There are two major mechanisms of Nrf2 transcription activation: depolymerization of Keap1 and Nrf2 under the stimulation of ROS or electrophilic reagents and direct phosphorylation of Nrf2 by PKC, mitogen-activated protein kinase (MAPK), and PI3K.25,26 These events occur during spermatogenesis but are not possible in late spermiogenesis or mature sperm. Defense systems exist in somatic cells but may be limited in sperm due to their limited pool of antioxidant proteins that cannot be replenished. Increased levels of Nrf2 in the sperm nuclei might provide insight that there may have been higher levels of ROS present during spermatogenesis, which damaged the cells, leading to lower semen quality.

In our study, significantly low Nrf2 and p-Nrf2 protein levels were found in the group with low sperm quality compared to those in the control group with normal sperm motility. According to previous research findings, Nrf2 is a cotranscription factor that regulates antioxidant enzymes and may protect sperm from the toxic effects of ROS and inflammation. We speculated that low sperm motility in patients with seminal vesiculitis was associated with the lower activity of Nrf2, indicating weaker anti-ROS and anti-inflammation functions. In addition, the lower protein levels of p-Nrf2 in the group with low sperm quality suggested that less Nrf2 transcription was activated through the direct phosphorylation of Nrf2. In a previous study, it was reported that low levels of the long noncoding RNA HOX antisense intergenic RNA (HOTAIR) are positively associated with Nrf2 levels in the spermatozoa of patients with asthenozoospermia or oligoasthenozoospermia.27 Chen et al.12 also found that Nrf2 mRNA levels are significantly lower in human males with low sperm motility. Our study was designed to identify whether Nrf2 plays a role in the pathogenesis of low sperm motility in patients with hematospermia caused by spermatocystitis. The sperm samples in our experimental group were collected from patients with seminal vesiculitis through transurethral seminal vesiculoscopy. In patients with spermatocystitis, sperm quality is affected by the seminal plasma, which may contain inflammatory cytokines. When exposed to inflammatory stimuli, sperm can increase the levels of cytoprotective enzyme-encoding genes, such as those in the Nrf2 signaling pathway. However, the persistent harmful effect of inflammatory cytokines on sperm may result in excessive activation of Nrf2 transcription. As a result, there were low Nrf2 protein levels in the group with spermatocystitis. In turn, the low Nrf2 protein levels suggested that the affected sperm were equipped with weaker anti-ROS and anti-inflammatory abilities. The significantly elevated levels of TNFα and IL-6 in the seminal plasma in the sperm samples of patients with persistent hematospermia in our study also suggested that antioxidant signaling involving Nrf2 and p-Nrf2 was elevated in the semen of the patients.

Overall, our results showed decreased Nrf2 and p-Nrf2 in sperm samples and increased TNFα, IL-6, and H₂O₂ levels in the seminal plasma of seminal vesiculitis patients compared to healthy volunteers. Patients with seminal vesiculitis also exhibited low sperm quality parameters. From the above analysis and the study results, it can be speculated that inflammatory changes in seminal vesicles causing a persistent harmful effect of inflammatory cytokines (TNFα and IL-6) and ROS (H₂O₂) on sperm may result in excessive consumption of activated Nrf2 along with decreased p-Nrf2 (the active form of Nrf2) and Nrf2. Therefore, decreased Nrf2 can be regarded as a biomarker of sperm damage by ROS and inflammatory cytokines in patients with seminal vesiculitis.

There were several limitations in our study. First, baseline differences among the participants could not be completely eliminated, which may result in bias in the measurement results. Second, we did not design in vitro experiments to identify the mechanism by which inflammatory cytokines or oxidation products degrade sperm function. Finally, our study had a small sample size.

In summary, the significant differences in Nrf2 and p-Nrf2 levels between spermatozoa samples from patients with persistent hematospermia and healthy volunteers suggest that Nrf2 signaling might play a role in the low sperm quality in patients with intractable spermatocystitis. Compared to healthy males, decreased Nrf2 can be regarded as a biomarker in sperm collected from patients with spermatocystitis through transurethral seminal vesiculoscopy.

AUTHOR CONTRIBUTIONS

SZW and JNL carried out project development, statistical analysis, and manuscript writing. FFZ, YJPW, and PZ carried out sample collection, data analysis, and results production. STC carried out study approval and manuscript writing. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.