Introduction

Non-gonococcal urethritis (NGU) is the most common disease of the lower urogenital tract in men [1]. Epidemiological data indicate a significant increase in the incidence of NGU over the past decades — from 50 million cases per year at the end of the last century to almost 90 million cases annually at the present time [2]. Inflammation in the urethra can lead to urethrogenic spread of infection with the development of pathology in the zone of the prostate-vesicular complex and the organs of the scrotum, which contributes to a decrease in the reproductive potential of men [3, 4]. In addition, NGU in men is often associated with inflammatory diseases of the pelvic organs in their female sexual partners and thus has an impact on women's reproductive health [5-7]. The medical and social significance of NGU is due to the fact that the disease occurs in predominantly young men, which, in turn, increases the risk of infection and spread of sexually transmitted infections (STIs), including HIV [8, 9].

Determining the causes of NGU development in some cases presents certain difficulties. One of the most difficult stages in the diagnosis is determining of the etiological factor for urethra inflammation, which directly affects the choice of an adequate etiotropic treatment and contributes to its effectiveness increase.

According to modern concepts, the generally accepted etiological agents of NGU are C. trachomatis, M. genitalium, and T. vaginalis [10]. Less common causes of NGU are herpes simplex viruses type 1 and 2, Epstein-Barr virus [11] and adenoviruses [12, 13], as well as U. urealyticum with a high concentration of bacteria in the locus [14, 15]. Among the possible reasons for urethra inflammation in men, a number of aerobic bacteria are considered — Haemophilus sp. [16, 17], Neisseria meningitidis [18, 19], Moraxella catarrhalis [20, 21], Streptococcus pneumoniae [22, 23], protozoa — Giardia lamblia, Entamoeba histolytica [24, 25], but quantity of controlled studies concerning the association of these microorganisms with NGU are few.

According to experts, after the exclusion of «recognized» pathogens related to STIs, the etiological factor of inflammation in the urethra cannot be found in more than 35% of cases [26]. In the literature, such urethritis is called pathogen-negative or idiopathic [26, 27]. They can be caused both by non-infectious causes — trauma to the urethra, allergic reactions, metabolic disorders, congestion (lat: accumulation — for example, fluid) in the pelvic organs [28], and previously unidentified microorganisms or agents which are currently not recognized as etiological.

Recent studies have shown the possibility of microflora exchange between sexual partners [29, 30], when microorganisms found in the lower parts of the reproductive tract in women can be considered as possible etiological agents for the urethra inflammation in the men.

One of the most common infectious diseases among women of reproductive age is bacterial vaginosis (BV). Its prevalence ranges from 10% to 30% in developed countries; and almost up to 70% in some regions of Africa [31—33]. BV is associated with an increased risk of perinatal complications, impaired women reproductive health and a negative impact on the life quality of patients [34—36].

We conducted a longitudinal study, including examination of sexual partners compared with control group in order to assess the clinical features of urethritis in men associated with BV in their sexual female-partners.

Material and methods

The study was conducted on the basis of the Moscow City Foundation for DNA Research from February 2016 to October 2018 as part of a prospective longitudinal study of outpatients who sought medical help at this institution. The study included 166 men aged 20 to 61 years (average age 34.81 ± 8.4 years) and 174 of their female sexual partners aged 19 to 69 years (average age 31.16 ± 7.74 years ). The study was approved by the local ethics committee. All patients included in the study gave written consent to participate in it.

The study inclusion criteria for patients were age over 18 years old, heterosexuality, and the absence of any treatment courses for 1 month or more. The criteria for exclusion from the study were refusal to participate in the study, severe concomitant somatic pathology, HIV infection.

After excluding the STIs, all men were divided into 2 observational groups: group 1 included patients with clinical and / or laboratory signs of urethritis, group 2 (control) — without urethritis. In each group, 2 subgroups were identified, depending on the presence or absence of BV in their sexual partners. Subgroup 1A consisted of patients with NGU and positive BV status of their sexual partners, subgroup 1B — patients with NGU and negative BV status of sexual partners.

Laboratory tests

The following clinical specimens were obtained from each male-patient:

1) discharge from the urethra for microscopic examination;

2) discharge from the urethra for molecular biological research;

3) a sample of the first portion of urine (FPU) for sediment microscopy and molecular biological research.

Samples of vaginal discharge were obtained from each female-patient for microscopic and molecular biological studies.

For microscopic examination of Gram-stained smears, samples were obtained using a sterile disposable swab: in men, by inserting an instrument into the urethra by 1–2 cm, in women after insertion of a vaginal speculum from the posterior or lateral fornix of the vagina, followed by distribution of the material in a thin layer over the surface slide.

For molecular biological research, the material was obtained by scraping with a sterile urogenital probe from the mucous membrane of the urethra in men and the vagina in women, followed by depositing the samples in sterile tubes with a transport medium with a mucolytic. Samples were stored at a temperature of 2 to 8° C until delivery to the laboratory of the Central Research Institute of Epidemiology of Rospotrebnadzor.

FPU samples for microscopy of the sediment and subsequent molecular biological study were obtained by self-collection of 7-10 ml of urine by male-participants in a sterile plastic container. A prerequisite for collecting material from male patients was urination delay for at least 4 hours before that.

To confirm the presence of inflammation in the men urethra, the results of microscopy of a smear from the urethra stained according to Gram at high magnification (×1000) were assessed with counting the number of polymorphonuclear leukocytes (PMNL) in an average of 5 visual fields. The diagnosis of urethritis was established when 5 PMNL or more were detected in the visual field. In addition, the confirmation of inflammation in the urethra was the detection of 10 PMNL or more in the visual field during microscopy of the FPU sediment at high magnification (×1000). The study of urine sediment allowed to increase the accuracy of microscopic assessment of the urethritis presence. When discordant results of microscopy of a smear from the urethra and FPU sediment were obtained, the diagnosis of urethritis was established with an increase in the number of PMNLs in at least one of the biomaterial types, while taking into account the presence of clinical signs of urethritis.

When the number of PMNLs in a smear from the urethra was from 5 to 20 in the visual field and in the sediment of PMNs from 10 to 40 in the visual field at high magnification (×1000), a conclusion was made about a low-grade inflammation/ process; if the number of PMNLs exceeded 20 visual field in a smear from the urethra and 40 in the PPM sediment, the severity of inflammation was regarded as highly pronounced (high-grade inflammation) [37].

DNA was extracted from samples of biological material for subsequent PCR studies for the presence of various microorganisms. DNA from biomaterial samples was extracted using the Amplisens kit "DNA-sorb AM" (Central Research Institute of Epidemiology) according to the attached instructions.

The causative agents of STIs (N. gonorrhoeae, C. trachomatis, M. genitalium, T. vaginalis, HSV 1, 2) were detected using PCR kits AmpliPrime-NCMT and AmpliSens HSV I, II-FL (Central Research Institute of Epidemiology). The results were calculated using the software supplied with the indicated reagent kits. Units of measuring the concentration of DNA of microorganisms were genomic equivalent in 1 ml of liquid (GE / ml).

The Amsel criteria were used to diagnose BV.

As part of the study, we developed an original patient questionnaire for men and women — participants in the study in order to register the passport data of patients, anamnestic data, risk factors for the development of inflammation in the lower parts of the urogenital tract, sexual history, patient complaints, clinical examination data and laboratory results in dynamics.

Statistical analysis of the data obtained was carried out on a personal computer using the MS EXCEL and IBM SPSS 23 application software packages. Distribution parameters (mean value, standard deviation, and frequency analysis) were calculated for all studied parameters in each group. The significance of differences between the study groups was assessed using Student's t-test. The indicators presented in the nominative scale were assessed using frequency analysis using Pearson's χ2 test. All obtained differences were considered significant at the level of significance not lower than p≤0.05.

Results

n 13 (7.8%) of 166 men initially included in the study, the examination revealed microorganisms, the etiological role of which in the development of urethritis is considered proven (N. gonorrhoeae, C. trachomatis, M. genitalium, T. vaginalis, HSV 1, 2). These patients, as well as their sexual partners, were excluded from the study. Thus, 153 men and 161 women continued to participate in the study. Men — participants were divided into 2 clinical groups in accordance with the results of the examination:

— Group 1 — men with clinical and / or laboratory signs of urethritis (n=94);

— Group 2, control — men without clinical and / or laboratory signs of urethritis (n=59).

In order to trace the possible relationship between the development of urethritis in men and the bacteria associated with BV in women, we assessed the state of the microflora of women — sexual partners of male in the study groups. Depending on the presence or absence of BV, the sexual partners of the men who took part in the study were assigned the status of BV-positive or BV-negative (Table 1).

Table 1. Distribution of patients by clinical groups depending on the presence of inflammation in the urethra and BV status of sexual partners

It was found that 46% of women — sexual partners of men in group 1 (men with urethritis) had the status of BV-positive. Moreover, in group 2 (control), only 22% of sexual partners were diagnosed with BV.

Thus, within each clinical group, 2 subgroups were identified, depending on the state of the microflora of sexual partners (presence or absence of BV).

Sociodemographic and behavioral characteristics of men

Among men with signs of inflammation in the urethra (group 1) and men in the control group (group 2), there were no significant differences in the level of education, monthly income, as well as in the average age of participants, approximately equal in both groups — 35.08 ± 8 , 12 and 35.3 ± 8.33 years (Table 2). However, it was noted that patients with urethritis and BV-positive status of sexual partners were on average 3.6 years younger than patients with urethritis, whose sexual partners had negative BV status (the average age of patients was 33.1 ± 7.74 and 36.7 ± 8.15, respectively). In addition, analysis of the distribution of men with inflammation in the urethra by age group depending on the BV status of sexual partners with a high level of reliability (p≤0.01) showed that 53.4% of patients with urethritis and positive BV status of sexual partners (subgroup 1A) were at the age of highest sexual activity (from 18 to 30 years). Only 25.4% of patients with negative BV-status of sexual partners belonged to this age group (subgroup 1B). The seeking medical help peak of patients in this group was noted in the age range of 31-45 years, at the same time, among patients with urethritis over 45 years old, BV-positive status of sexual partners was established more than 2 times less than BV-negative.

Table 2. Sociodemographic and behavioral characteristics of men

Sociodemographic and behavioral characteristics of men — participants in the study are presented in Table 2.

There were no significant differences among men of different clinical groups in the presence of STIs in the anamnesis, however, the study of the sexual history showed that men with a positive BV status of their sexual partners significantly more often (p≤0.01) had 2 or more sexual partners in the last 6 months than in men with negative BV-status of their sexual partners and healthy men in the control group — 60.5, 47.1 and 30.5%, respectively. At the same time, the practice of unprotected anal contacts with a high level of reliability was more often (p≤0.01) indicated by patients with urethritis and negative BV-status of their sexual partners (subgroup 1B).

Only 7% of men in subgroup 1A had no foreskin, while in 27.4% of patients in subgroup 1B and 22% of patients in group 2, it was preserved (p≤0.05).

Clinical manifestations of urethritis

Patient-assessed urethritis symptoms

An analysis of the clinical symptoms of urethritis in men in clinical groups is presented in Table 3.

Table 3. Comparative analysis of clinical symptoms of urethritis in men in clinical groups

As follows from the data given in Table 3, patients with urethritis and positive BV status of their sexual partners compared with patients with negative BV status of partners significantly more often (p≤0.05) did not notice discharge from the urethra, whereas in patients with inflammation in the urethra, whose sexual partners were BV-negative, with a high degree of certainty more often (p≤0.05), complained about the presence of abundant discharge from the urethra. Such subjective signs of urethritis as itching and burning in the urethra are less pronounced in patients of subgroup 1A, complaints of dysuria and pain in the lower abdomen and scrotal organs were significantly less frequent (p≤0.01) in this group compared with patients in subgroup 1B (a total of 4 (9.2%) and 25 (48.9%), respectively). In general, the absence of any subjective signs of inflammation in the urethra with a high degree of reliability was more often (p≤0.01) indicated by patients with urethritis and positive BV status of their sexual partners than patients with negative BV status of partners.

Objective clinical signs of urethritis

Clinical signs of urethritis identified during the examination of patients are presented in Table 4.

Table 4. Clinical signs of urethritis

Assessing the objective signs of urethritis, presented in Table 4, it can be concluded that in patients of subgroup 1A significantly more often than in subgroup 1B (p≤0.05), discharge from the urethra was of a mucous nature, while in patients of subgroup 1B, mucopurulent and purulent discharge prevailed. In addition, it was found that during the clinical examination of patients, scanty discharge from the urethra with a high degree of reliability was more often (p≤0.01) noted in subgroup 1A, and abundant discharge was significantly more often (p≤0.01) observed in patients of subgroup 1B. Assessment of objective signs of urethral inflammation also showed that hyperemia and edema of the urethral sponges were less frequently observed in patients of subgroup 1A compared with patients in subgroup 1B (69.7 and 80.3%, respectively).

Laboratory signs of urethritis in the examined men

Laboratory signs of urethritis, identified when evaluating the results of microscopy, are presented in Table 5.

Table 5. The severity of urethritis depending on the BV-status of sexual partners

From the data Table 5 it follows that in patients with a positive BV status of their sexual partners, a high grade of urethritis was noted significantly less frequently (p≤0.01) than in patients whose sexual partners had a negative BV status..

Discussion

According to modern data, the main etiological factors in men with NGU are pathogens related to STIs, however, after their exclusion in more than 35% of cases, it is not possible to establish the etiological factor of inflammation in the urethra [26, 38]. Obviously, disease directly affects the choice of adequate etiotropic treatment. In this regard, many researchers urge to avoid empirical treatment of urethritis whenever possible, in order not only to increase the effectiveness of treatment, but also to reduce the risk of developing microorganism resistance to antibiotics. [39—41].

In the literature devoted to the problems of NGU, the possibility of bacteria associated with BV in women to cause inflammation in the urethra in men has been discussed for a long time [42–47]. There is no certainty in this issue [12, 48, 49], therefore our research is very relevant.

Analysis of the data obtained in our study showed that women — sexual partners of men with urethritis, were diagnosed with BV more than 2 times often than in sexual partners of men in the control group. This fact supports the hypothesis of the possible etiological role of BV-associated microorganisms in the development of inflammation in the urethra in men, and the revealed reliably significant association of urethritis in patients with BV-positive status of sexual partners with the age of highest sexual activity, as well as with the presence of 2 sexual partners and more over the past 6 months provides additional arguments regarding the sexually transmitted nature of the origin of urethritis in these patients, since the above signs are considered to be one of the most important risk factors for STIs. Moreover, the possibility of exchange of BV-associated microorganisms between sexual partners during sexual intercourse is quite obvious and confirmed in a number of studies in recent years using nucleic acid amplification methods [29, 30, 50].

Our study showed that patients with positive BV status of their sexual partners were significantly more likely than patients whose sexual partners had negative BV status, as well as with patients in the control group, that the foreskin was preserved. Recent studies using molecular biological diagnostic methods have shown that bacteria associated with BV in women can be part of both the urethral and penile microbiota of men [30, 50, 51], while in men with preserved in the foreskin, the microbiota of the coronal sulcus was more heterogeneous than in circumcised males and was present in this locus in a higher concentration; anaerobic microorganisms prevailed [52, 53]. We hypothesized that a higher concentration of anaerobic microflora in the penile microbiota in men with preserved foreskin promotes more frequent penetration (autoinoculation) of this microflora into the distal urethra with the potential for inflammation of the urethral mucosa.

We noted that men with NGU and BV-positive status of their partners were significantly less likely to complain of dysuria and pain in the lower abdomen and scrotal organs than patients whose sexual partners had negative BV-status. According to experts, such complaints may indicate an ascending nature of the inflammatory process with damage to the posterior urethra and accessory gonads [54]. In this regard, it can be assumed that BV-associated microorganisms have a less aggressive potential and are localized mainly in the distal urethra in men. It is generally accepted that urethritis in men is usually accompanied by symptoms such as abnormal discharge, itching, burning in the urethra, and dysuria. It is the presence of these symptoms in most cases that is the most common reason for patients seeking medical help. At the same time, it is known that with NGU, inflammation in the urethra can have minimal clinical manifestations of the disease, and in some cases it can be completely asymptomatic for patients. In such cases, the diagnosis is made on the basis of laboratory data (an increase in the number of PMNLs with microscopy of a smear from the urethra, stained according to Gram and / or FPU sediment). The absence of subjective symptoms of the disease can lead to late seeking medical help and, as a consequence, to the chronicity of the inflammatory process with the subsequent risk of complications, often accompanied by a decrease in reproductive function in both men and their female sexual partners. [55—57].

Evaluating the data of laboratory diagnostics of urethritis, obtained in the course of this study, we can note the revealed fact of a significantly lower severity of the inflammatory reaction in the urethra in men with a positive BV-status of sexual partners in comparison with similar data obtained in patients whose sexual partners were BV-negative status, which correlates with the less pronounced clinical manifestations of urethritis described above in patients of this group. Analyzing these observations, it is possible to draw a parallel between the effect of BV-associated microorganisms on the mucous membranes of the lower parts of the reproductive tract of women and men. On the one hand, it is known that women with BV do not have a leukocyte reaction from the vaginal mucosa, and among the Amsel criteria for diagnosing BV there is no indication of an increase in the number of leukocytes in a vaginal smear [58]. Perhaps, this can explain the significantly lower severity of inflammation in the urethra caused by BV-associated bacteria in men. However, on the other hand, the very fact of detecting leukocytes in the urethra in men in a situation where the presence of the same microorganisms in the vagina of partners did not entail the development of an inflammatory reaction is of considerable interest to us and requires further understanding.

Obviously, among the possible reasons for the development of inflammation in the urethra in men, one can consider other opportunistic microorganisms, the diagnosis of which is not included in our study (EBV, adenoviruses, Haemophilus sp., Neisseria meningitidis, etc.), as well as previously not recognized microorganisms, to identify which many researchers are working on improving methods for amplifying nucleic acids and developing special molecular biological tests [59, 60].

Considering the possible reasons for the development of urethritis in men who took part in the study, it should be noted that there is a more frequent practice of unprotected anal intercourse in patients of subgroup 1B compared with subgroup 1A and the control group, which may indicate a possible role of saprophytic and opportunistic intestinal flora in the development of inflammation in the urethra in men whose sexual partners were BV-negative.

The limitation of our study was also the impossibility of differential diagnosis of urethritis of infectious and non-infectious etiology. However, the totality of the observations may indicate the etiological role of microorganisms responsible for the development of BV in women in the occurrence of inflammation in the urethra in men.

Distribution of men with urethritis by age categories depending on the BV status of sexual partners.

Conclusions

1. Urethritis in men, associated with BV in their sexual partners, can be considered as an independent disease, the characteristic distinguishing features of which are the relatively low severity of inflammation in the urethra, as well as the paucity of clinical symptoms and signs of the disease.

2. The prevalence of men at the most sexually active age among patients with BV-associated urethritis and signs of risky sexual behavior confirm the hypothesis about the possibility of transmission of BV-associated microorganisms during sexual intercourse.

3. Circumcision reduces the risk of BV-associated urethritis in men.

For a more complete assessment of the data obtained, it is planned to analyze the microbiological status of the urogenital biotope among patients of the selected clinical groups, as well as to assess the clinical and microbiological effectiveness of treatment aimed at eradication of BV-associated microorganisms in the case of confirmation of their exclusively presence in the urethra in men with inflammation in the urethra.

Authors’ contributions:

The concept and design of the study: Gomberg M.A., Kim D.G., Guschin A.E.

Collecting and interpreting the data: Kim D.G.

Drafting the manuscript: Gomberg M.A., Kim D.G., Guschin A.E.

Revising the manuscript: Gomberg M.A., Guschin A.E.

The authors declare no conflict of interest.