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Volume 24, Issue 3
March 2017
Pages 211–216
Original Article: Clinical Investigation

Seminal microbiome in men with and without prostatitis


Authors
Reet Mändar,
Margus Punab,
Paul Korrovits,
Silver Türk,
Kristo Ausmees,
Eleri Lapp,
Jens-Konrad Preem,
Kristjan Oopkaup,
Andres Salumets,
Jaak Truu
First published:
1 February 2017Full publication history
DOI:
10.1111/iju.13286 View/save citation
Cited by (CrossRef):
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Article has an altmetric score of 9
Funding Information
Abstract

Objectives

To profile the seminal microbiome applying next generation sequencing.
Methods

Semen samples of 67 men were involved in the study (21 men with and 46 men without prostatitis). Seminal microbiomes were profiled applying the method that uses combinatorial sequence tags attached to polymerase chain reaction primers that amplify the
ribosomal ribonucleic acid V6 region. Amplified polymerase chain reaction products were sequenced using an Illumina paired-end protocol on HiSeq2000 platform.
Results

The most abundant phylum in semen was Firmicutes, comprising nearly half of the sequences found (median 41.7%, quartiles 28.5–47.2%) followed by Bacteroidetes, Proteobacteria and Actinobacteria. The counts of lactobacilli were higher in healthy men
than prostatitis patients (27% [20.2–34.6%] vs 20.2% [4.9–25.0%]; P = 0.05), especially for Lactobacillus iners. Proteobacteria comprised higher proportions in prostatitis patients than healthy men. The species richness was higher in prostatitis
patients than healthy men (inverted Simpson index 13.5 ± 5.8 vs 10.3 ± 4.0). Conclusions

The semen of chronic prostatitis patients contains fewer health-supporting lactobacilli, and has higher species diversity than that of healthy men. Firmicutes (especially lactobacilli), Bacteroidetes, Proteobacteria and Actinobacteria comprise the
highest proportion of seminal microbiome.
Abbreviations & Acronyms
NGS
next-generation sequencing
NIH
National Institutes of Health
rRNA
ribosomal ribonucleic acid
WBC
white blood cells
WHO
World Health Organization
Introduction

Prostatitis is a common but poorly understood condition that could affect up to half of men of all ages and demographics, and its prevalence is approximately 10% in the population of middle-aged men.[1] Different forms of the disease have been organized
into NIH Prostatitis Classification (Table S1). The etiology of acute bacterial and chronic bacterial prostatitis (categories NIH I and NIH II) involves acknowledged urinary tract pathogens. At the same time, the remaining categories are far more
frequent, but their etiology has remained largely unclear.[2, 3]
The most widespread category of prostatitis – Chronic Prostatitis/Chronic Pelvic Pain Syndrome (NIH III) is divided to inflammatory (NIH IIIA) and non-inflammatory (NIH IIIB) subtypes. To this date, no single pathomechanism has been proved yet, but an
interrelated multifactorial cascade has been proposed where an initiating event (infection, trauma, etc) might lead to immunological and neurogenic stimulation, inflammation, neuropathic damage with afferent nerve upregulation, and ultimately, pain.[4]
Asymptomatic inflammatory prostatitis (NIH IV) is usually found by chance.[5] The problems with NIH III and IV prostatitis include inadequate understanding of etiology and pathogenesis, insufficient methods for diagnosing, as well as deficient treatment schemes. In addition to subjective discomfort, prostatitis is associated with
reduced semen quality, including negative effect on sperm concentration, motility, vitality and morphology.[6] Furthermore, there is an overlap between prostatitis syndrome and several other diseases, such as male accessory gland infection, interstitial
cystitis, benign prostate hypertrophy, irritable bowel syndrome and others.[7] The treatment is best done using multiple simultaneous therapies aimed at the different aspects of the condition, including alpha-blockers, psychological intervention and
prostate-directed therapy.[3] Though the etiology remains unclear, there is a longtime habit of treating prostatitis patients with antibiotics. This approach is supported by some studies that have involved advanced diagnosing methods rather than routine
cultures, and that have shown differences in genital tract microbial communities between prostatitis patients and healthy men. Anaerobic bacteria, difficult-to-culture or wall-deficient bacteria and bacteria that exist in biofilms within obstructed ducts
in the prostate can be important in the etiology of chronic prostatitis.[8-10] The NGS studies of microbial communities in the prostate-specific specimens of prostatitis patients are missing so far.
We aimed to compare the seminal microbiome in men with and without chronic prostatitis applying Illumina sequencing.
Methods

Study group

The study was carried out at Tartu University Hospital (Tartu, Estonia) and Competence Center on Health Technologies (Tartu, Estonia) in 2010–2013.
The study group included 67 men participating in a prospective study of the male genital tract microbiome (Fig. S1). They consulted the physician for prostatitis complaints, for fertility status check or for prophylactic purposes. The prostatitis
complaints were recorded using the NIH-Chronic Prostatitis Symptom Index questionnaire, and the inflammatory reaction in their semen was recorded using cytological analysis (Table S1). If an inflammatory reaction was found in the semen of asymptomatic
men, then they were included into the NIH IV category of prostatitis patients. As the result, in 21 men, inflammatory (NIH IIIA or NIH IV category, n = 10) or non-inflammatory (NIH IIIB category, n = 11) prostatitis was diagnosed. The men without pelvic
pain/discomfort complaints and leukocytospermia were included into the control group.
Exclusion criteria for all men were stated according to the suggestions of the NIH workshop on chronic prostatitis and included other urogenital tract infections.[11] None of the men had received antimicrobial therapy within 3 months. The mean age was 33.
3 years in prostatitis patients and 28.8 years in controls. Clinical data of the study participants are presented in Table S2.
Ethics statement

Participation in the study was voluntary. Written informed consent was obtained from all study participants. The study was approved by the Ethics Review Committee on Human Research of Tartu University, Tartu, Estonia (protocol no. 193/T-16, 31.05.2010).
Specimens

Samples were collected after washing the glans penis with soap and water, and after urinating. Semen was obtained by masturbation, ejaculated into a sterile collection tube and incubated at 37°C for 25–45 min for liquefaction. Basic semen parameters
and leukocytospermia were detected immediately. For NGS, the samples were stored at −80°C before DNA extraction.
Characterization of semen

The analysis of semen was carried out according to WHO guidelines.[12] Semen volume was estimated by weighing the collection tube with the semen sample and subsequently subtracting the predetermined weight of the empty tube assuming 1 g = 1 mL. Motility
was assessed in order to report the number of motile spermatozoa (WHO motility classes A + B). Sperm concentration was assessed using the Neubauer hemocytometers (Merck KGaA, Darmstadt, Germany). Total sperm count was calculated by multiplying semen
volume by sperm concentration. A qualified laboratory technician carried out all semen analyses. These data are presented in Table S2.
Cytological analysis of semen

Semen smears were made for detecting WBC. The smears were air-dried, Bryan–Leishman stained and examined with the use of oil immersion microscopy (magnification: ×1000) by an experienced microscopist. The WBC concentration in semen was calculated by
using the known sperm concentration (as 106/mL) according to the following formula:
display math
A total of 100 round cells were counted twice, and their mean value was registered.
Illumina sequencing

Semen samples were characterized by profiling the microbial community on the basis of the 16S rRNA gene by amplifying the bacterial-specific V6 hypervariable region of the 16S rRNA gene. Details of the sequencing are provided in Table S3.
Statistical analysis

The linear discriminant analysis effect size algorithm and Mann–Whitney rank sum test were applied to assess the differences between prostatitis patients and controls. Spearman's rank order correlation was used to find associations between the
parameters. For the 16S rDNA amplicon sequencing data, principal coordinate analysis was used to explore and visualize similarities between seminal and vaginal microbiota samples based on the obtained Yue and Clayton measure of dissimilarity.
Results

Altogether 67 men were investigated, among them were 21 men with chronic prostatitis (10 men with inflammatory and 12 men with non-inflammatory prostatitis) and 46 controls. Clinical data of the study participants are presented in Table S2.
Microbiome of semen

The bacterial diversity in semen samples was characterized by sequencing the V6 region of 16S rRNA genes. The species diversity was higher in prostatitis patients than healthy men (inverted Simpson index 13.5 ± 5.8 vs 10.3 ± 4.0).
The most abundant phylum in the semen was Firmicutes, comprising nearly half of the sequences found (median 41.7%, quartiles 28.5–47.2%). The other numerous phyla included Bacteroidetes (19.5% [14.8–26.8%]), Proteobacteria (15.9% [12.7–24.2%]) and
Actinobacteria (9.7% [7.1–13.0%]), whereas other phyla comprised less than the hundredth part of an average community (including Fusobacteria, Spirochaetes, Synergistetes, Tenericutes, Acidobacteria, Cyanobacteria and others).
The most abundant genera included Lactobacillus, Gillisia, Prevotella, Corynebacterium and Gardnerella (Table 1). The most abundant species included Lactobacillus iners, Lactobacillus crispatus, Gardnerella vaginalis and Corynebacterium seminale.
Correlations between the frequent species are given in Table S4, and correlations between clinical parameters and bacteria in Table S5. The two Lactobacillus species were in strong association with each other, and with vaginal bacteria (Gardnerella,
Mobiluncus, Atopobium, Varibaculum) and Bacteroides ureolyticus. Corynbacterium seminale and other coryneform bacteria were in association with Gram-positive aerobic and anaerobic cocci. Vaginal bacteria (including bacterial vaginosis related bacteria)
appeared to be associated with younger age, and environmental bacteria with older age.
Table 1. Most frequent genera and species in the semen samples (mean proportions are given)
Healthy (%) Prostatitis (%) Total (%)
a P < 0.05 between patients and controls.
b P = 0.078 between patients and controls.
Most frequent genera
Lactobacillus 25.2 19.0 23.3
Gillisia 9.8 11.1 10.2
Prevotella 7.4 4.9 6.6
Corynebacterium 4.3 6.6 5.0
Gardnerella 4.5 3.0 4.0
Dechloromonas 3.4 4.4 3.7
Diaphorobacter 2.5 2.9 2.6
Finegoldia 1.9 2.9 2.2
Porphyromonas 2.2 1.8 2.1
Peptoniphilus 1.6 2.4 1.9
Dialister 1.8 1.4 1.7
Curvibacter 1.5 1.9 1.6
Anaerococcus 1.2 1.6 1.3
Campylobacter 0.8 1.6 1.1
Atopobium 1.1 0.6 1.0
Pseudomonas 0.8 1.1 0.9
Flavobacterium 0.8 0.9 0.8
Staphylococcus 0.9 0.7 0.8
Veillonella 0.8 0.7 0.8
Streptococcus 0.6 0.8 0.7
Acidovorax 0.6 0.9 0.7
Enterobacter 0.6 0.6 0.6
Acinetobacter 0.4 0.4 0.4
Varibaculum 0.4 0.5 0.4
Peptostreptococcus 0.4 0.3 0.4
Janthinobacterium 0.4 0.5 0.4
Shuttleworthia 0.4 0.2 0.4
Anaerovorax 0.2 0.4 0.3
Actinomyces 0.2 0.3 0.3
Fusobacterium 0.3 0.2 0.2
Dysgonomonas 0.2 0.3 0.2
Mobiluncus 0.2 0.2 0.2
Aggregatibacter 0.1 0.2 0.1
Bifidobacterium 0.1 0.1 0.1
Most frequent species
Lactobacillus iners a 13.4 9.1 12.0
Lactobacillus crispatus 10.0 8.0 9.4
Gardnerella vaginalis a 4.5 3.0 4.0
Corynebacterium seminale b 2.1 4.6 2.9
Peptoniphilus asaccharolyticus 1.3 1.7 1.5
Atopobium vaginae a 1.1 0.6 1.0
Enterobacter cowanii 0.6 0.6 0.6
Pseudomonas veronii 0.5 0.6 0.6
Campylobacter rectus 0.3 1.2 0.5
Bacteroides ureolyticus 0.6 0.4 0.5
Anaerococcus hydrogenalis 0.5 0.7 0.5
Streptococcus infantis 0.4 0.5 0.4
Acinetobacter johnsonii 0.4 0.4 0.4
Varibaculum cambriense 0.4 0.5 0.4
Peptostreptococcus anaerobius 0.4 0.3 0.4
Janthinobacterium lividum 0.4 0.5 0.4
Seminal microbiome in prostatitis patients and controls

Figure S2 shows the clustering of microbial community data in different participants groups, showing that the communities of control participants are more uniform than prostatitis patients.
The most remarkable difference between the groups appeared in the counts of lactobacilli (Fig. 1, Table 1) that were higher in healthy men than prostatitis patients (median 27% [quartiles 20.2–34.6%] vs 20.2% [4.9–25.0%]; P = 0.05), especially on
behalf of Lactobacillus iners (14.2% [8.8–19.4%] vs 9.8% [3.2–14.3%]; P = 0.013). Proteobacteria comprised higher proportions in prostatitis patients than healthy men – 19.6% (15.2–27.7%) versus 15.5% (12.0–21.5%); however, this difference did
not reach the significance level.
Figure 1.
Figure 1.
Open in figure viewer
Linear discriminant analysis effect size analysis results. (a) Histogram of the l inear discriminant analysis scores computed for differentially abundant bacterial taxa between prostatitis patients and controls. (b) Taxonomic representation of
statistically significant differences between semen microbiomes of healthy men and prostatitis patients. Differences are represented in the color of the most abundant classes (red indicating prostatitis, green healthy and yellow non-significant). Each
circle's diameter is proportional to the taxon's abundance.
Discussion

In the present study, we obtained semen samples from healthy men and chronic prostatitis patients, and sequenced the 16S rRNA gene fragments using a NGS platform. The present study revealed polymicrobial communities in the semen samples, the most
prevalent phyla were Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria. Major differences between the prostatitis patients and controls appeared to be lower species diversity and higher counts of lactobacilli in healthy men than prostatitis
patients. According to our best understanding, this is the first study investigating semen microbiome in prostatitis patients applying the Illumina platform.
To date, the microbiome of semen has been studied applying advanced molecular methods, mostly in connection with male infertility. There are few studies that have used molecular methods before the NGS era in which a wide spectrum of bacteria in semen has
been detected.[13, 14] In both studies, high numbers of anaerobic bacteria were found. Some studies have recently used the NGS method to detect bacteria in semen.[15-17] Weng et al. investigated the male partners of infertile couples, and found that the
proportions of Lactobacillus and Gardnerella were higher in the normal samples, whereas that of Prevotella in the low-quality samples.[15] Hou et al. found a negative association between sperm quality and the presence of Anaerococcus, while also many
vaginal bacterial taxa were found in their study.[16] At the same time, the results of Liu et al. differ from the two aforementioned studies where abundant lactobacilli were found – Streptococcus, Corynebacterium and Staphylococcus were found to be
common semen bacteria in men who have sex with men.[17] These studies point out the impact of partner's microbiome on the male reproductive tract communities. In a recent study, Shoskes et al. investigated urinary microbiome of prostatitis patients, and
found that 17 clades were overrepresented (e.g. Clostridia), and five were underrepresented (e.g. Bacilli) in prostatitis patients.[18]
In our present study, we found polymicrobial communities from the semen samples where the phylum Firmicutes comprised nearly half of the sequences found. The list of most frequent genera generally coincided with the data of previous studies, except for
genus Gillisia – a Gram-negative anaerobic rod with low enzymatic activity. Our most important finding was scarcity of health-supporting lactic acid bacteria in prostatitis patients in comparison with healthy controls. This difference appeared mostly
at the expense of L. iners. Lactobacilli are commonly present in the human vagina, oral cavity and gastrointestinal tract, where they possess a significant protective function. As their proportion in semen was remarkably high according to the present
study, as well as some other studies, a similar function might be attributed to them in the male genital tract.[16, 19] Semen quality in Lactobacillus-predominant semen samples is higher than in the case of the other community types,[15] and lactobacilli
prevent sperm lipid peroxidation, thus preserving sperm motility and viability.[20]
It is interesting to note that our previous culture-based studies have not revealed lactobacilli in semen samples, although we used suitable media (MRS agar) for them. Our present study applying NGS showed remarkable proportions of lactobacilli, the
majority of them being L. iners – the smallest Lactobacillus discovered to date that has also an unusually small genome. The strain's genome encodes several genes that allow it to respond adequately to rapid changes in the environment. Due to special
nutrient requirements, it does not grow on common lactobacillus media, and little is known about its characteristics. L. iners is a common constituent of the vaginal microbiota[21, 22] while recently it has been found also in the male genital tract.[15,
19]
We also found that Proteobacteria comprised higher proportions in prostatitis patients than healthy men, although this difference did not reach the significance level. Gram-negative rods belonging to this phylum are acknowledged urinary tract pathogens
causing also acute and chronic bacterial prostatitis (NIH categories I and II).[23]
We showed some correlations between the frequent species (Table S4). The two Lactobacillus species were in strong association with each other, and with vaginal bacteria (Gardnerella, Mobiluncus, Atopobium, Varibaculum). Bacteroides ureolyticus that has
been associated with toxic effects towards spermatozoa, but might be associated with bacterial vaginosis, was also associated with this complex.[24] Corynbacterium seminale and other coryneform bacteria that are frequently present in the male genital
tract appeared to be in association with Gram-positive aerobic and anaerobic cocci.
Regarding the associations between bacteria and clinical parameters (Table S5), we surprisingly showed a high number of correlations between age and bacteria – vaginal bacteria appeared to be associated with younger age, and environmental bacteria with
older age, which can be explained with the different sexual activity rate. This confirms the results of our previous study showing a high concordance between semen and vaginal samples within a couple.[25] Bacteria that were associated with lower sperm
quality did not form a clear pattern; however, many Gram-negative anaerobes were involved.
In the case of any prostate-specific material, there is an inherent possibility of urethral contamination, as numerous aerobic, microaerophilic and anaerobic bacteria belong to the microbiota of the urethra.[26] Our research team has compared the
communities in semen with that of first-catch urine, which reveals urethral microbiota.[27] The microorganisms’ concentration and the number of species were significantly higher in patients’ semen than in patients’ urine, and these specimens shared
only one-third of species, showing that most of the semen microorganisms in prostatitis patients originate from the upper genital tract.
Another related question is the possible contamination during NGS studies. Several genera of Proteobacteria, such as Pseudomonas, Curvibacter and Oxalobacter that were found in the present study, have been termed as contaminants [Ref. j in Table S3].
However, these bacteria were not found in the mock community, and the counts of Proteobacteria tended to be higher in the prostatitis group.
Our second remarkable finding was the higher species diversity in prostatitis patients than healthy men. Hence, we could confirm the results obtained by our previous culture-based studies where the main difference between chronic prostatitis patients and
controls was quantitative – prostatitis patients harbored significantly higher total bacterial concentration and higher number of different species in their semen than controls.27–29 Prostatitis is a complicated clinical entity, and opinions about
the plausibility of its infectious etiology have ranged from one extreme to another.[9] The present data suggest that it might be viewed as an unfavorable shift in the balance of genital tract microbiota. Our findings fit to the hypothesis that
dysbacteriosis in the male genital tract could be related to chronic prostatitis.[30] Whether and when the usual commensals of the male genital tract might behave as pathogens is an unanswered question so far. In some former studies, the prostatitis-
related male genital tract bacteria have shown higher biofilm production[31] and anticomplement activity[32] than the bacteria isolated from healthy men. Prostatitis might be similar to several other endogenous infections where no single pathogen, but a
set of species, is involved. Although there is little specificity as to the involvement of single species, specificity becomes more apparent when bacterial community profiles are taken into account. The concept of the community as pathogen is based on
the principle of teamwork.[33] The behavior of the community and the outcome of the host/bacterial community co-work will depend on the species composing the community, and how the associations that can occur within the community affect and modulate the
virulence of involved species. Bacteria that individually might have low virulence and are unable to cause disease can do so when in association with others.
A limitation of the present study was the small size of the study group, and the median age of the healthy participants was slightly younger that that of the patients. As we used semen as specimens, we could not eliminate the urethral bacteria; however,
they form the minority of seminal microbiota, as shown previously.[27] Another limitation was that the 16S rRNA gene fragments sequenced were too short to reliably identify all the sequences to the species level. Furthermore, the viability of
microorganisms detected by NGS is unclear. However, the present study still gave some interesting results.
In conclusion, semen of chronic prostatitis patients contains less health-supporting lactobacilli and has higher species diversity than that of healthy men. Firmicutes (especially lactobacilli), Bacteroidetes, Proteobacteria and Actinobacteria comprise
the highest proportion of seminal microbiome. This is the first study comparing seminal microbiome in chronic prostatitis patients and healthy men applying Illumina sequencing.
Acknowledgments

This study was supported by the Estonian Research Council (grant no. IUT34-19 and PUT181), Estonian Ministry of Education and Research (grant no. KOGU-HUMB), University of Tartu (grant no. SARMBARENG) and Enterprise Estonia (grant no. EU48695).
Conflict of interest

None declared.
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