Introduction

Infertility, defined as the inability to achieve pregnancy after 1 year of regular intercourse [1], affects 15 % of couples in reproductive age. About 10–20 % of the inability to achieve pregnancy can be explained by male infertility and 30–40 % is explained by both male and female infertility [2]. Many factors can cause male infertility, and approximately 15 % of male infertility is associated with the infection in the genital tract [3]. U. urealyticum and M. hominis are commonly found in the genital tract of patients experiencing symptoms including infertility, orchitis, epididymitis, prostatitis, and nongonococcal urethritis [4, 5] as well as in asymptomatic subjects [6, 7].

For about a decade, U. urealyticum and M. hominis infections have been recognized as a common sexually transmitted disease (STD) in developed countries [8]. These microorganisms naturally inhabit the male urethra and contaminate the semen during ejaculation. However, both these microorganisms, particularly U. urealyticum, are potential pathogens that play etiologic roles in both genital infections and male infertility [9, 10]. Several studies have analyzed the relationship between U. urealyticum and M. hominis infections and semen quality, and some have demonstrated that these infections alter various characteristics of semen, such as sperm motility, density, and morphology, and that antibiotic treatment can improve the semen quality [11, 12]. On the contrary, other researchers did not find that U. urealyticum and M. hominis infections affect either semen quality or male infertility [13]. Contrary to the evidence that these microorganisms play an important role in the pathogenesis of human infertility, no convincing causal relationships between the infections and human infertility have yet been established.

In the present study, we examined the prevalence of U. urealyticum and M. hominis infections in infertile and fertile men and their effect on semen quality by analyzing the morphology, semen volume, pH value, sperm concentration, progressive motility, and total motility. Furthermore, the drug resistance of these microorganisms was evaluated.

Materials and methods

Study population

Ethics approval from the regional ethics committee has been granted for the focus groups with infertile and fertile men. A total of 19,098 men of infertile couples who visited the Reproductive Center, the Reproductive and Genetic Hospital of CITIC, Xiangya, China, from January to December 2014 were enrolled in this study. The patients were enrolled for semen analysis following a failure to impregnate their wives after at least 1 year of unprotected sexual intercourse. Past history of infections and sexual history were obtained from all patients. The following exclusion criteria were applied: (1) reproductive system abnormalities, known hereditary and/or familial disorders; (2) heavy alcohol use (>60 g/day); (3) heavy smoking (>20 cigarettes/day); and (4) exposure to physical or chemical agents with known negative reproductive effects. The control group included 3368 fertile men who donated sperm at the sperm bank from January 2011 to December 2014. Men whose semen parameters were normal and/or those whose wives had nonassisted pregnancies in the past were considered fertile.

Ureaplasma urealyticum and M. hominis detection and antimicrobial susceptibility testing

Mycoplasma IST (Crest, China) was used for the detection and antimicrobial susceptibility testing of both microbial species. Urethral samples were fed on lyophilized medium to diagnose U. urealyticum and M. hominis infections and drug susceptibilities. The samples were homogenized by slight rotation. Then, 100 µL of the samples was transferred to microplates containing antibiotics and proliferation wells separately for the qualitative detection of U. urealyticum and M. hominis. One drop of sterile mineral oil was added to each well to generate anaerobic conditions for culture. Microplates were closed with caps and incubated at 37 °C for 24 and 48 h, respectively. For detection, U. urealyticum and M. hominis growth could be detected using the indicator phenol red in the medium. Both strains were tested for susceptibility to thiamphenicol, doxycycline, erythromycin, azithromycin, clarithromycin, josamycin, levofloxacin, ciprofloxacin, roxithromycin, and gatifloxacin.

Semen collection

Semen samples were collected by masturbation into a sterile container after 3–5 days of abstinence. The samples were liquefied at 37 °C for about 30 min in an incubator before analysis.

Semen analysis

The semen was analyzed using the methods outlined by the World Health Organization (WHO, 5th edition), and the volume, pH, progressive motility (PR), total motility (PR + NP), sperm concentration, normal forms, and total motile sperm count (TMC) were determined.

Statistical analysis

SPSS 19.0 statistical software was used for statistical analysis. The results are reported as mean ± standard deviation unless otherwise indicated. The t test and chi-square test were used to compare continuous variables and categorical variables, respectively. A statistically significant difference was defined when the P value was <0.05.

Results

Demographic characteristics

A total of 19,098 semen samples were taken from patients aged 23–44 years (28.6 ± 7.2), individuals in the fertile group were those who were aged 20–35 years (26.9 ± 5.5), no differences were found in the age between fertile and infertile groups (P = 0.16), and the groups did not differ significantly in frequency of sexual intercourse, abstinence times, BMI, alcohol drinking, and smoking (Table 1).

Table 1 Demographic characteristics of participants
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Prevalence of U. urealyticum and M. hominis in infertile and fertile men

The prevalence of these pathogens significantly differed between infertile and fertile men (Table 2). The prevalence of U. urealyticum-positive specimens among urethral specimens in infertile and fertile men was 10.22 % (1951/19,098) and 3.65 % (123/3368; χ 2, 147.203; P < 0.001), respectively. M. hominis was detected in 3.16 % (604/19,098) and 0.89 % (37/3368) of infertile and fertile men, respectively (χ 2 test, 41.490; P < 0.001). Finally, 1.8 (343/19,098) and 0.48 % (16/3368) of fertile men were found to harbor mixed pathogens (χ 2, 31.771; P < 0.001).

Table 2 Prevalence of Ureaplasma urealyticum and Mycoplasma hominis infections in infertile and fertile men
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Effects of U. urealyticum and M. hominis on semen quality

The semen variables in the infertile and fertile men are summarized in Table 3. The mean values of sperm concentration, progressive motility, total motility, normal forms, and TMC were significantly lower in infertile men than in fertile men. To identify the effects of U. urealyticum and M. hominis on semen, we compared semen parameters of the infected and uninfected subjects. Some of the parameters including sperm concentration, progressive motility, total motility, normal forms, and TMC tended to be lower in the infected group than in the uninfected group, although the differences were not significant in the case of M. hominis-infected and uninfected individuals (P > 0.05; Table 4). The progressive motility, total motility, and normal forms showed significant difference between the U. urealyticum-infected, mixed infection, and uninfected groups (P < 0.05). The TMC was significantly lower in the mixed infection group (P = 0.017) than in the uninfected group.

Table 3 Comparison of seminal parameters in infertile and fertile men
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Table 4 Effects (mean ± SD) of Ureaplasma urealyticum and Mycoplasma hominis infections on seminal variables in infertile men
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Drug susceptibility test results

Table 5 presents the antibiotic resistance profiles of each microorganism. As shown in the table, 1857 (95.2 %) and 1851 (94.9 %) of the 1951 patients with U. urealyticum infection were susceptible to doxycycline and josamycin, respectively. Furthermore, 1709 (87.6 %) of the 1951 patients with U. urealyticum infection were resistant to levofloxacin, and 1826 (93.6 %) were resistant to ciprofloxacin. A higher proportion of patients (330/343; 96.2 %) with both U. urealyticum and M. hominis showed higher resistance to ciprofloxacin at higher critical concentrations. Mixed infections were more susceptible to doxycycline and josamycin than either U. urealyticum or M. hominis infection alone. Mixed infections were more resistant to ciprofloxacin.

Table 5 Susceptibility of U. urealyticum and M. hominis to ten different antibiotics
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Discussion

The relationship between U. urealyticum and M. hominis infection and male infertility has been studied widely; however, the results remain controversial. Most patients with these infections are not aware of their infections because they do not experience any symptoms. These inconsistencies are probably because of small sample sizes and confounding factors, such as social and economic factors and sexual activity. Meanwhile, different populations have different susceptibility, which may be also one of the reasons for this controversy. China is one of the most populous countries in the world. The rapid pace of economic and social change in China over the past two decades has been accompanied by an increased rate of infertility [14, 15]. In addition, antibiotic abuse will lead to drug resistance. As a result, the treatment options for U. urealyticum and M. hominis infections are becoming more and more limited. Our study illustrates an important link between the presence of these microorganisms and infertility in men and provides guidance for reasonable use of antibiotics in China.

Although there is considerable disagreement on the exact association of U. urealyticum and M. hominis with male infertility, it is generally agreed that the prevalence of both these infections is higher in infertile men than in fertile men [16–19]. Our study demonstrated a statistically significant higher detection rate of U. urealyticum in the urethral specimens from infertile men (10.22 %) compared to fertile men (3.65 %) and of M. hominis in the urethral specimens from infertile men (3.16 %) compared to fertile men (0.89 %). The detection rates for U. urealyticum and M. hominis in infertile men were approximately threefold higher than the corresponding rates in fertile men. U. urealyticum is the smallest free-living organism that most commonly inhabits the urogenital tract and has been found in male seminal fluids with a prevalence ranging from 10 to 42 % [20, 21]. The high rate of U. urealyticum detection in this study suggests that it is widespread among infertile men, which is consistent with previous findings [16, 17]. The prevalence of M. hominis varies significantly between infertile and fertile men and was low in our study; other studies have also found that M. hominis is uncommon in the urethra of men [22]. We were concerned that technical problems and detection method might have resulted in the low prevalence observed in this study. However, our results were in agreement with the findings reported previously, suggesting that the prevalence of M. hominis in the urethra of the male Chinese population is indeed low. In our study, U. urealyticum and M. hominis were often detected in both infertile and fertile men, suggesting that they may have a commensal relationship.

In addition to studying the prevalence of these microorganisms in infertile and fertile men, we also assessed the relationship of these microorganisms with sperm quality. The semen quality is considered to be one of the most significant indicators of male fertility. Previous studies on the effects of U. urealyticum and M. hominis infections or colonization on semen parameters also show conflicting results. Some studies have reported that these microorganisms have no real effect on the quality of semen [23, 24], but others have demonstrated associations between U. urealyticum and M. hominis and semen quality [10, 25]. In this study, we evaluated the correlation between progressive motility, total motility, and normal forms and U. urealyticum infection in infertile men. Mixed infection of U. urealyticum and M. hominis was found to be associated with lower progressive motility, total motility, normal forms, and TMC. However, although M. hominis infection was associated with a reduction in motility and sperm concentration, we failed to demonstrate any significant correlation between the M. hominis infection and semen quality (P > 0.05). The results of this study are consistent with previous findings [24, 26]. These microorganisms may diminish fertility by adhering to spermatozoa and directly altering sperm morphology, as well as motility and survival [24].

The susceptibilities of genital mycoplasmas to antimicrobial agents differ by geographic region [27]. The difference in the antimicrobial resistance found in reports from various countries might be due to the differences in the guidelines of antimicrobial usage. Our result showed that both U. urealyticum and M. hominis had relatively low resistance to josamycin and doxycycline in China. Similar results were found in recent studies by Yang [28], Chen [29], and Yang [30]. Therefore, josamycin and doxycycline should be the primary choice for U. urealyticum and M. hominis in China.

This study has some limitations. First, the study did not include leukocytes as a variable in the semen parameters. The presence of leukocytes in the semen may be indicative of infection or inflammation; however, there remains controversy about the significance of true leukocytospermia. The role of leukocytes in semen in predicting reproductive performance warrants further study. Second, although the semen volume, pH value, sperm concentration, and other parameters were studied, we did not study the changes in these parameters following antibiotic treatment and we also did not study the pregnancy rates before and after antibiotic treatment; studying the post-treatment changes in these parameters and pregnancy rates may better support our findings. Third, the donated sperm will be chosen for the patients with azoospermia and severe oligozoospermia, but we still included them in the study because the proportion of the patients with azoospermia and severe oligozoospermia was small, and they may still have infections.

At last, U. urealyticum and M. hominis infections may adversely affect the seminal quality in both infertile and fertile men; however, we did not study the seminal quality of fertile men because this study focused primarily on the effects of U. urealyticum and M. hominis infections on the seminal quality of infertile men. Future studies could study the seminal quality of both fertile and infertile men with these infections.

In conclusion, this study revealed that U. urealyticum and M. hominis were widely prevalent in the male population, both infertile and fertile, in China. Our results revealed that U. urealyticum, but not M. hominis, can negatively influence the seminal quality. Therefore, clinicians should pay close attention to these infections, particularly U. urealyticum infection, in infertile men. Our antibiotic susceptibility test results indicate that doxycycline and josamycin should be the primary choice in the empirical treatment of these infections.