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From the Urological Research Institute of PLA, Southwest Hospital, Third Military Medical University, Chongqing, China.
| Correspondence to: Dr Bo Song, Urological Research Institute of PLA, Southwest Hospital, Third Military Medical University, Chongqing, Ch China 400038 (e-mail: songbo7951{at}163.com). |
| Received for publication March 16, 2009; accepted for publication September 21, 2009. |
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Abstract |
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Key words: Semen, calcification, urine
lu et al,
2003), urolithiasis
(Ciftçiolu et al,
2005), malignant microcalcifications in breast cancer
(Altundag et al, 2006), aortic
valve calcification (Bratos-Pérez et
al, 2008), and human arthritic synovial fluid
(Tsurumoto et al, 2008). In this prospective study, we determined whether NB participate in the pathogenesis of TM. Semen and urine samples after pretreatment were cultured according to the culture methods for NB. The acquired microbes from the culture flasks were identified by indirect immunofluorescent staining (IIFS), transmission electron microscopy (TEM), and 16s rRNA gene expression.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The procedures of sample collection, including urine and semen, from 34 subjects were under strict aseptic conditions. A portion of the urine and semen specimens cultured on tryptic soy agar plates was used to detect whether patients had bacterial infections; if a patient was found to be positive for bacterial infection, he was excluded from further study. The remaining urine and semen samples were used for NB culture.
Culture of NB
According to the culture techniques of Ciftçiolu and
Kajander (Ciftçiolu and
Kajander, 1998; Kajander and
Ciftçiolu, 1998), semen and urine samples were
pretreated with oscillation and 1:3 dilution in a sterile glass with sterile
water. Then the samples were demineralized by adding 1 M HCl, which was
subsequently neutralized with 1 M NaOH. The resultant was filtered (pinhole
filter, 0.45 µm and 0.22 µm; Millex; Millipore Carrigtwohill, Cork,
Ireland) and centrifuged at 20 000 x g for 45 minutes, after
which the supernatant fluids were removed. The remnant samples were cultured
in flasks containing RPMI 1640 (Invitrogen, Carlsbad, California) with 10%
gamma-irradiated fetal bovine serum (Sigma-Aldrich, St Louis, Missouri) and
kept at 37°C (pH 7.4) in 5% carbon dioxide/95% air. Sterile normal saline
was cultured as the negative control.
Identification of Cultured Microbes
Smears made of isolated culture pathogenic bacteria for IIFS were air-dried
and fixed in methanol (–20°C) before blocking with 2% goat serum
phosphate-buffered saline (PBS). The sections were incubated in a humidified
chamber for 24 hours with mouse monoclonal antibody 8D10 against NB (NanoBac
Oy, Kuopio, Finland) used at a 1:10 dilution, washed in PBS, and then treated
with a 1:500 dilution of tetramethyl rhodamine isothiocyanate–labeled
goat anti-mouse IgG (Chemicon, Temecula, California) for 1 hour.
4',6-diamidino-2-phenylindole dichloride was used to label the nucleus.
PBS was used to replace the 8D10 antibody as the negative control. A Zeiss LSM
510 meta confocal laser scanning microscope (CLSM; Carl Zeiss, Jena, Germany)
was used for IIFS-CLSM.
For TEM, the microbes were placed on 200 mesh copper grids, negatively stained with 2% phosphotungstic acid for 20 to 30 seconds, and then evaluated by TEM.
16s rRNA Gene Expression
Genomic DNA was acquired from NB cultures using the FastDNA spin kit (BIO
101, Vista, California), and 16s rRNA gene sequences of NB were amplified
using the following primers: A, 5'-AACGAACGCTGGCGGCAGGC-3' and B,
5'-CACCCCAGTCGCTGACCC-3'. After the amplified fragment was
purified with a gel extraction kit (Viogene, Taipei, Taiwan), it was ligated
into the pGEM-T vector (Promega, Madison, Wisconsin) to transform ECOS
competent cells (Yeastern Biotech, Taipei, Taiwan). The inserted fragment in
the recombinant plasmid was sequenced using the following primers: TAF,
5'-CAAGGCGATTAAGTTGGGTA-3' and TAR, 5'-GGAATTGTGAGCGATAA
CA-3' and then compared with homologous gene sequences in the GenBank
database using the BLAST tool.
Statistical Methods
All data are expressed as means ± SD. The 
2 test and
Student's t test were performed using SPSS statistical software
(version 16.0; SPSS Inc, Chicago, Illinois). P < .05 was
considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Identification of Cultured Microbes
NB are difficult to stain with common dyes. Mouse monoclonal antibody 8D10
against NB has been successfully used for the detection and identification of
NB (Ciftçiolu and Kajander,
1998; Kajander and
Ciftçiolu, 1998). In this study, IIFS-CLSM showed
that the cultured microbes were clustered with different sizes and about 100
to 500 nm in diameter (Figure
1). TEM revealed that the microbes were spheroid with a black coat
and crystals around the bacterial body
(Figure 2). These distinctive
features coincided with those of NB described in other studies
(Ciftçiolu and Kajander,
1998; Kajander and
Ciftçiolu, 1998). From these results, we concluded
that the cultured microbes from semen samples of the infertile patients were
NB.
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16s rRNA Gene Expression
NB have a 16s rRNA gene sequence (European Molecular Biology Laboratory
database, X98418 and X98419), and they belong to the alpha-2 subgroup of
Proteobacteria based upon that sequence
(Ciftçiolu and Kajander,
1998). The alpha-2 subgroup of Proteobacteria contains bacteria
that are able to penetrate eukaryotic cells. Analysis of the 16s rRNA gene
sequence has been successfully used for the identification of NB and their
pathogenicity (Kajander and
Ciftçiolu, 1998;
Wood and Shoskes, 2006). In
this study, we obtained the target sequence length of 1400 bp after DNA
sequencing for the inserted fragment in the recombinant plasmid. According to
the GenBank database, the BLAST result showed that the observed sequence was
97% similar to the 16s rRNA gene of NB (GenBank accession number, X98419) with
a score of 2480, identity 97%, and E-value of 0, which implied that the
cultured microbes were NB and could be causative agents.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Whether NB could be a cause of TM and participate in the formation of TM
may be a worthy topic in need of discussion. NB are novel Gram-negative
microorganisms with small sizes (100–500 nm) that are able to form
calcium phosphate crystals at neutral pH
(Miller et al, 2004). There is
evidence supporting the notion that NB could be causative agents. For example,
they are able to grow and exert cytotoxic effects
(Hjelle et al, 2000;
Ciftçiolu et al,
2007). Now NB have been found in periodontal diseases
(Ciftçiolu et al,
2003; Demir,
2008), urolithiasis
(Ciftçiolu et al,
2005), aortic valve calcification
(Bratos-Pérez et al,
2008), and human arthritic synovial fluid
(Tsurumoto et al, 2008) and
have been proven to participate in the clinical pathologic processes of those
diseases. In this study, we successfully isolated and cultured NB from semen
samples of infertile patients with TM, which have been found to be composed of
calcium phosphate and degenerating intratubular cells in the seminiferous
tubules (De Jong et al, 2004).
Based on the above data, we hypothesized that NB may participate in the
pathogenesis of TM, particularly in infertility. Apatite produced by NB may
play a key role in the formation of TM by making a central calcium phosphate
deposit around which other components are collected. In this study, we could
not confirm where NB came from with the available data. In vivo, NB are voided
mainly through the urinary system and have been isolated within the
genitourinary tract, including in polycystic kidney disease, renal calculi,
and chronic prostatitis (Wood et al, 2006). Therefore, we infer that NB in
urine may reflux into the seminiferous tubules and survive there. Further
studies are needed to elucidate the potential mechanism by which NB originate
in infertile patients with TM.
The role of TM in producing symptoms and disease is controversial,
particularly because of the high prevalence in some asymptomatic men. However,
there is a clear correlation between TM and infertility. As with seminiferous
tubule calculi, the key factor in causing infertility is obstruction, which
may bring about secondary inflammation, increase intraseminiferous pressure,
and affect the testes blood supply. Such a mechanism could explain why
antibiotic procedures may provide improvement in sperm quality. However,
therapy does not always enhance the probability of conception
(Dohle et al, 2005). NB are
self-propagating calcifying macromolecular complexes and are resistant to most
antibiotics. No other bacteria are as resistant to elimination as NB
(Ciftçiolu et al,
2003), which are best inhibited by tetracycline
(Miller et al, 2004).
Therefore, conventional clinical treatments have not been able to eradicate
NB. Inflammation and development of calcification caused by NB in the area of
the seminiferous tubules could persistently affect sperm quality and cause
infertility.
Color Doppler ultrasonography has become a valuable method for diagnosing scrotal abnormalities in the infertile population (Sakamoto et al, 2006; Qublan et al, 2007), which increases the frequency of diagnosing TM. In infertile patients undergoing scrotal ultrasonography, the incidence of TM varies from 1.5% to 2.8% (Ganem et al, 1999; Qublan et al, 2007). In this study, 17 patients were found to have TM from 832 infertile patients (2.0%), which coincides with the above incidence. However, our results (Table) indicated that the incidence of epididymal cysts in the TM group was a bit higher than in the control group. We suggest that NB in the TM group may cause some cases of epididymal cysts, and further study about the association between NB and epididymal cysts will be needed. Other studies have shown an association between testicular tumors and TM (von Eckardstein et al, 2001; Ringdahl et al, 2004; Miller et al, 2007). All patients enrolled in our study were informed about monthly self-examinations. Patients also received physical examination and testicular tumor marker evaluation at 3-month intervals and scrotal ultrasonography at 6-month intervals in the follow-up period.
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Although we detected NB in semen samples of infertile patients with TM in this study, a clear relationship between NB and infertility with TM could not be drawn from our data. Hence, more work needs to be done in this area. Specifically, anti-NB treatment for infertile patients with TM is recommended. Our hypothesis, if proven with evidence-based clinical trials, may have significant impact on the pathogenesis and treatment for those infertile patients with TM.
Conclusions
Self-replicating calcific nanometer-scale particles, similar to those
described as NB from other calcific human tissues, can be cultured and
identified from semen samples of infertile patients with TM. These data
implicate a possible link between the presence of NB and the development of
TM. However, whether NB contribute to the pathogenesis of the disease or are
only innocent bystanders needs to be clarified in further studies.
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Acknowledgments |
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Footnotes |
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