Abstract
Objectives
The present study aimed to investigate the effects of Ureaplasma urealyticum (U. urealyticum, UU) infection on sperm DNA, semen parameters and sperm quality in male patients for interpreting the proposed mechanisms of male infertility.
Methods
The morphology of sperm was observed under the standard bright-field microscope at 1000× magnification after the hematoxylin-eosin (HE) staining. All the experiments of semen biochemical parameters of seminal plasma zinc, fructose, citric acid, neutral α-glucosidase, elastase, and sperm acrosin activity were performed on the SK6000 Fully Automated Biochemical Analyzer. The values of DNA fragmentation index (DFI) and high DNA stainability (HDS) in semen were tested by using the sperm chromatin structure assay (SCSA).
Results
The seminal plasma elastase level increased (p = 0.0006) and sperm morphology abnormality increased in UU-positive group (p < 0.0001). There was no significant difference in each of other semen parameters including semen volume, leukocyte concentration, seminal plasma zinc, fructose, citric acid, neutral α-glucosidase, and sperm acrosin activity (p > 0.05 for each). The values of DFI in the UU-positive group were higher than those in the UU-negative group (p < 0.01), but there was no significant difference in HDS between UU-positive group and UU-negative group (p > 0.05).
Conclusions
UU infection in male genital tract can not only significantly increase the seminal plasma elastase levels and sperm morphological abnormalities, but also markedly elevate sperm DNA fragmentation levels and decrease sperm DNA integrity. It may represent an important pathway contributing to male infertility.
Keywords: Ureaplasma urealyticum, Infection, Sperm, DNA, Quality
Introduction
Ureaplasma species are a genus of microorganism including two human-associated species: Ureaplasma urealyticum (U. urealyticum, UU) and Ureaplasma parvum (U. parvum, UP). Currently, approximately 15% couples of reproductive age experience infertility globally [1], and male-factor infertility accounts for about a half of all infertile cases [2, 3]. Many studies seem to support the role of UU infection in the genital tract in male infertility, whereas there is little evidence suggesting an association between UP and compromised male fertility [4–6]. Besides, a few reports announced no association of both UU and UP infections in the genital tract with male infertility [7]. Therefore, the precise role of either UU or UP infection in the genital tract in male infertility remains controversial.
Up to now, the data on mechanisms of UU infection in the genital tract leading to male infertility are still limited [8–10]. Most of the studies on this topic have seemed to indicate that UU infection in the genital tract can decrease sperm quality primarily by inducing semen inflammation, causing sperm malformation and sperm DNA impairment [11–13].
It is well known that male accessory sex glands secrete several factors such as zinc, fructose, citric acid, and neutral α-glucosidase which are crucial for sperm physiology. Zinc acts as a regulator of prostate metabolism, with its concentration in the prostate being significantly higher than in serum or other bodily fluids. It also plays a critical role in protecting sperm DNA integrity and maintaining motility [14, 15]. Similarly, citric acid in seminal plasma is almost entirely secreted by the prostate gland, making it a highly specific biochemical marker for prostate function [16]. Seminal plasma fructose serves as a characteristic biochemical marker for seminal vesicle function and is also a crucial energy source for sperm metabolism. Neutral α-glucosidase in seminal plasma is primarily secreted by the epididymis and serves as a characteristic enzyme of the epididymis, reflecting its functional status [17]. Impaired secretory function of the male accessory glands can lead to poor semen quality. Infections in these glands may further suppress the production of key secretory markers, although this effect is often temporary [17]. Besides, seminal plasma elastase produced by neutrophils or immune cells is a marker of genital tract infection or chronic inflammation. Sperm acrosin is an essential product synthesized by the sperm acrosome itself for normal fertilization and is used to evaluate sperm function and fertility. However, the data about the impact of infection on the function of the human male accessory sex glands are very scarce and remain controversial [18–21]. It remains unclear whether and how UU infection in the genital tract impairs human male accessory sex gland function.
Hence, the current study was intended to evaluate the role of UU infection in the genital tract on male infertility and explore the routes of UU infection in the genital tract leading to male infertility.
Materials and methods
Ethics statement
This study has been approved by ethics committee of the The Affiliated Taian City Central Hospital of Qingdao University (Approval No., QDU-HEC-2022135). All patient data were anonymously reported, with no possibility of connecting the positive specimens with individual patients. In the present study, individual patient data were not collected and additional informed consent was therefore not required.
Patients and clinical specimens
A total of 335 male patients with genitourinary symptoms were enrolled in this investigation and research in The Affiliated Tai’an City Central Hospital of Qingdao University from July 2023 to May 2025. The genital tract secretions were collected from these patients first, immediately followed by semen collection. Male genital tract secretions were collected by inserting sterile cotton-tipped swabs into the urethra approximately 2–4 cm and rotating for 3–5 s. Semen specimens were collected by ejaculating through masturbation after 2–7 days of sexual abstinence into sterile standard containers. Semen specimens that failed to liquefy or exhibited incomplete liquefaction were excluded, and only completely liquefied semen samples were analyzed for semen parameters. Male genital tract secretions or semen specimens were sent immediately to Clinical Microbiology Laboratory for culture. In the culture method, a sample was considered UU-positive if the phenol red indicator turned from yellow to red, and UU-negative if there was no color change. Based on the results of culture method, patient population were divided into UU-positive (n = 150) and UU-negative (n = 185) groups. Genomic DNA was subsequently extracted from the remaining broth culture material for molecular analysis. This molecular analysis was performed to verify the results of the culture method. Then the broth culture materials remains were stored at − 80 °C together with the remained genomic materials for later use.
Culture of UU in clinical specimens
Culture and identification of UU isolates were achieved by using a commercially available Mycoplasma ICS Test Kit (Livzon Diagnostics Inc., Zhuhai, China). After 24 h of broth culturing at 37 °C, the identification of UU isolates was realized by an increase in pH from pH6.5 to pH.8.0 due to urea hydrolysis in the medium that can change the phenol red indicator from yellow to a red.
Molecular detection of UU
Genomic DNA was extracted from UU broth culture remains described above by UNIQ-10 column type bacterial DNA Isolation Kit (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer’s instructions.
UU was detected by a commercially available TaqMan Real-time Quantitative PCR Detection Kit (DAAN Gene Co., Ltd. of Sun Yat-sen University, Zhongshan, China) according to the manufacturer’s instructions. Experiments were run on the ABI Quant Studio 5 Real-time PCR instrument (Thermo Fisher Scientific, USA). Amplification reactions were carried out as follows: initial denaturation at 93 °C for 2 min, then 10 cycles of denaturation at 93 °C for 45 s and annealing at 55 °C for 60 s, and finally 30 cycles of denaturation at 93 °C for 30 s and annealing at 55 °C for 45 s. Negative control and a series of dilutions positive standards from 1.0 × 108 to 1.0 × 104 copies/mL in the Detection Kit were run along with samples in each run. Samples are considered positive for UU if the threshold cycle is less than 30 according to the manufacturer’s instructions.
Testing of seminal volume, leukocyte concentration, and sperm morphology in semen samples
Freshly collected semen was immediately incubated at 37 °C in an incubator before analysis for 15–60 min for liquefaction. After liquefaction of the ejaculate, the seminal volume was measured with a graduated pipette and the leukocytes in semen were determined by using the o-toluidine-based peroxidase staining method according to the “WHO laboratory manual for the examination and processing of human semen” (Sixth Edition, 2021) [17].
The morphology of sperm was observed under the standard bright-field microscope (Olympus CX21, Olympus Corporation, Tokyo, Japan) at 1000× magnification after the hematoxylin-eosin (HE) staining. The sperm morphological examination was carried out by counting at least 200 spermatozoa or whole slides (if less than 200 sperm) per sample with double-blinded method. Next, morphological abnormalities of sperm organelles (nucleus, acrosome and tail) were evaluated, and the percentage of sperm with ideal morphology was calculated.
Detection of seminal plasma zinc, fructose, citric acid, neutral α-glucosidase, elastase, and sperm acrosin activity
Seminal plasma zinc was analyzed using the PAR (4-(2-Pyridylazo) resorcinol) assay. Seminal plasma fructose, citric acid, and α-glucosidase were determined by hexokinase assay, citrate lyase assay, and PNPG (p-Nitrophenyl-α-D-glucopyranoside) colorimetric assay, respectively. Seminal plasma elastase was detected via enzyme-linked immunosorbent assay (ELISA). Besides, Sperm acrosin activity was measured by the BAPNA (Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride) assay. All the experiments were performed on the SK6000 Fully Automated Biochemical Analyzer (Sinothinker Technology Co., Ltd., Shenzhen, China) and the results were interpreted strictly following the manufacturer’s instructions. According to the Detection kit instruction, the reference values for semen biochemical parameter in fertile men are listed as below: Seminal plasma zinc ≥ 2.4 µmol/ejaculation; Seminal plasma fructose ≥ 13 µmol/ejaculation; Seminal plasma citric acid ≥ 52 µmol/ejaculation; Seminal plasma neutral α-glucosidase ≥ 20 mU/ejaculation; Seminal plasma elastase < 290 ng/mL; and Sperm acrosin activity ≥ 59.3 µIU/10⁶ sperm.
Detection of DFI and HDS in semen specimens
In clinical practice, sperm DNA impairment includes sperm DNA fragmentation and sperm chromatin condensation abnormality. Sperm DNA fragmentation is commonly expressed using the DFI, while sperm chromatin condensation abnormality indicating sperm immaturity is typically characterized by HDS. The values of DFI and HDS in semen were tested by using the sperm chromatin structure assay (SCSA) on a Sparrow AISFCM Flow cytometry (Celula Biotechnology Inc., Chengdu, China) according to the manufacturer’s instructions. Test results were interpreted according to the kit instruction with the criteria listed below: A DFI value of ≥ 30% is considered as poor sperm DNA integrity, while a DFI value between > 15% and < 30% is classified as fair sperm DNA integrity. A DFI value of ≤ 15% is considered as high sperm DNA integrity. As for HDS, a HDS value of > 15% is considered as abnormal sperm chromatin condensation, while a HDS value of ≤ 15% is considered as normal mature sperm chromatin content.
Statistical analysis
The results of semen parameters were reported as mean ± standard deviation or percentage (%). The t test and chi-square test were used to compare differences of semen parameters between UU-positive group and UU-negative group by using IBM SPSS Statistics 28.0 statistical software (SPSS Inc., Chicago, IL, USA). A p-value of less than 0.5 was considered statistically significant.
Results
Effects of UU infection on semen parameters in semen specimens
The comparison of these semen parameters was made between the UU-positive group and the UU-negative group. The comparative results demonstrated that normal sperm morphology in the UU-positive group (3.18% ± 1.70%) was much lower than that in the UU-negative group (4.90% ± 2.12%, t=−8.24, p < 0.0001). Moreover, the seminal plasma elastase level indicating the status of inflammation in the UU-positive group (95.48 ± 81.42) was much higher than that in the UU-negative group (66.87 ± 66.12, t = 3.47, p = 0.0006) (Table 1). However, there was no statistically difference in the other seminal parameters including semen volume, leukocytes, seminal plasma zinc, fructose, citric acid, neutral α-glucosidase, and sperm acrosin activity between the UU-positive group and the UU-negative group (p > 0.05 for each, Table 1).
Table 1.
Comparison of semen parameters between UU-positive group and UU-negative group in Tai’an, China
| Semen parameters | UU-positive group (Mean ± SD) (n = 150) | UU-negative group (Mean ± SD) (n = 185) | t value | p value |
|---|---|---|---|---|
| Semen Volume (mL) | 3.57 ± 1.57 | 3.39 ± 1.33 | 1.12 | 0.265 |
| Leukocyte concentration (×10⁶/mL) | 0.38 ± 0.94 | 0.28 ± 0.23 | 1.27 | 0.205 |
| Normal sperm morphology (%) | 3.18 ± 1.70 | 4.90 ± 2.12 | −8.24 | < 0.0001 |
| Zinc (µmol/ejaculation) | 8.57 ± 5.75 | 8.42 ± 5.24 | 0.25 | 0.805 |
| Fructose (µmol/ejaculation) | 42.07 ± 24.53 | 40.52 ± 28.99 | 0.53 | 0.596 |
| Citric acid (µmol/ejaculation) | 93.06 ± 53.81 | 96.51 ± 49.66 | −0.60 | 0.546 |
| Neutral α-glucosidase (mU/ejaculation) | 51.90 ± 56.51 | 48.93 ± 29.68 | 0.58 | 0.561 |
| Elastase (ng/mL) | 95.48 ± 81.42 | 66.87 ± 66.12 | 3.47 | 0.0006 |
| Sperm acrosin activity (µIU/10⁶ sperm) | 112.95 ± 39.52 | 108.68 ± 39.52 | 0.98 | 0.326 |
Effects of UU infection on DFI and HDS
In the UU-positive group, the percentage of patients with poor sperm DNA integrity (DFI values ≥ 30%) was much higher than that in the UU-negative group (17.33% verse 1.62%, χ²= 25.86, p < 0.0001). Moreover, the percentage of patients with fair sperm DNA integrity (DFI values between > 15% and < 30%) in UU-positive group (42.67%) was higher than that in UU-negative group (25.41%, χ²=11.14, p = 0.0084). However, although the percentage (8.67%) of patients with abnormal mature sperm chromatin content (HDS values > 15%) indicating abnormal sperm chromatin condensation in the UU positive group was numerically higher than that in the UU negative group (5.41%), but the difference did not reach statistical significance (χ²=1.38, p = 0.24) (Table 2).
Table 2.
Comparison of DFI and HDS between UU positive group and UU negative group in Tai'an, China
| Sperm Impairment Markers | UU positive group (n=150) | UU negative group (n=185) | χ² | p value | |||
|---|---|---|---|---|---|---|---|
| Number | Percentage (%) | Number | Percentage (%) | ||||
| DFI | ≥30 | 26 | 17.33 | 3 | 1.62 | 25.86 | <0.0001 |
| 15-30 | 64 | 17.33 | 47 | 25.41 | 11.14 | 0.0084 | |
| HDS | >15 | 13 | 8.67 | 10 | 5.41 | 1.38 | 0.24 |
Discussion
Currently, there are a limited number of reports studying the correlation between the seminal plasma elastase levels and sperm characteristics. The seminal plasma elastase may be a reliable marker that can reflect the inflammatory status of the genital tract [22–24]. The current study has shown that UU infection in the genital tract can increase greatly the seminal plasma elastase levels in the semen samples. Furthermore, this study has demonstrated that UU infection in the genital tract can increase dramatically the sperm morphology abnormality, consistent with the previous studies [8, 11]. However, the current study has shown that UU infection in the genital tract can not decrease the semen volume and increase the leukocyte concentration in semen specimens, contrary to the previous studies [8, 11, 25, 26]. The lack of the association between UU-Infection and semen volume might be associated with the lack of the connection of UU-infection with fructose level as a marker for seminal vesicle function, since the seminal vesicle contribute mainly to semen volume. Additionally, the lack of the association between UU-Infection and leukocytes shows the poor diagnostic value of leukocytes in the detection of inflammation in the male genital tract, as previously discussed by Punab et al. [27] and Aghazarian et al. [28]. This finding indicates a strong correlation between UU infection in the genital tract and decreased normal sperm morphology. This result shows that UU-infection exerts its deleterious effect on sperm cells at testicular level, as sperm morphology is determined in the testis during the process of spermiogenesis. This finding also indicates a strong correlation between UU infection in the genital tract and increased seminal plasma elastase levels in the semen samples, thereby representing important route contributing to male infertility.
In this study, a comparative analysis between the UU-positive group and UU-negative group has shown that UU infection in the genital tract can not induce significant alterations of semen biochemical parameters including seminal plasma zinc, fructose, citric acid, neutral α-glucosidase, and sperm acrosin activity (p >0.05 for each) (Table 2). This indicates that UU infection in the genital tract can not apparently impair the function of the human male accessory sex glands, contrary to the previous studies [25, 26]. This conclusion depends on which stage UU is detected in the genital tract. In the preliminary stages of infection, we can not observe the effect of UU on the male accessory glands. By ascending infection, the male accessory glands can be affected, thus resulting in epididymitis, seminal vesiculitis or prostatitis, potentially leading to conclusions that would be otherwise.
Notably, our observations in the current study have shown that the DFI values in the UU-positive group was significantly higher than that in the UU-negative group (p < 0.01), indicating a strong correlation between UU infection and decreased sperm DNA integrity. This is another evidence that the impairment of sperm quality by UU occurred in the testis, as nuclear development is also completed during the process of spermiogenesis. This finding aligns with a report from Xi’an city of China [29], but differs from previous reports which failed to find any association between UU infection and the impairment of sperm DNA integrity [12, 30]. When it comes to HDS, there is no significant difference in HDS between the UU-positive and UU-negative groups, suggesting that UU infection is not associated with reduced sperm DNA maturation, contrary to the previous reports [12, 31].
It is well known that many patients with genital UU infections in various countries exhibit subtle symptoms or asymptomatic [9, 30]. This kind of persistent asymptomatic colonization or chronic inflammation in the genital tract caused by UU may not cause apparent damage on the function of the human male accessory sex glands in the preliminary stages of infection. However, this asymptomatic persistent colonization or chronic inflammation can not only significantly increase the seminal plasma elastase levels and sperm morphological abnormalities [8, 11], but also markedly elevate sperm DNA fragmentation levels and decrease sperm DNA integrity [29]. It may substantially reduce sperm quality and represent an important pathway contributing to male infertility. Furthermore, this type of asymptomatic persistent colonization or chronic inflammation poses an even greater harm, because it can cause substantial damage to human reproductive health unnoticedly [32]. The limitations of this study include: (1) the absence of data for several basic semen parameters, including total sperm count, motility, and vitality; and (2) the lack of measurements for reactive oxygen species (ROS) and proinflammatory cytokines, such as IL-6 and IL-8.
Conclusion
In summary, UU infection in male genital tract can not only significantly increase the seminal plasma elastase levels and sperm morphological abnormalities, but also markedly elevate sperm DNA fragmentation levels and decrease sperm DNA integrity. It may represent an important pathway contributing to male infertility.
Acknowledgements
AcknowledgmentsThis work was supported by the grant from Natural Science Foundation of Shandong Province (ZR2023MH301). The authors thank the whole staff of the Clinical Microbiology Laboratory, Reproductive Medicine Laboratory, and Molecular Biology Laboratory of our hospital for their help regarding the laboratory operations.
Author contributions
Lihong Zhao: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing–original draft, Writing–review & editing. Yuanyuan Jiang: Data curation, Formal analysis, Investigation, Methodology, Resources, Supervision, Validation. Yulong Zong: Data curation, Formal analysis, Methodology, Resources, Software, Supervision. Ruiying Li: Data curation, Validation, Investigation, Resources, Supervision, Visualization. Hui Zhang: Data curation, Investigation, Methodology, Resources. Jin Wang: Methodology, Resources, Software. Zhe Li: Data curation, Investigation,, Resources. Shuping Zhao: Conceptualization, Funding acquisition, Project administration. All authors have read and agreed to the published version of the manuscript.
Data availability
No datasets were generated or analysed during the current study.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
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Associated Data
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Data Availability Statement
No datasets were generated or analysed during the current study.