Chapter

The effect of genital tract

17 infection and inflammation on
male infertility
Sanjay S. Kasturi, E. Charles Osterberg, Justin Tannir, and Robert E. Brannigan

Introduction Subclinical infections are more difficult to recog-

Infection and inflammation of the male reproductive nize and diagnose than acute infections. This is true
tract are complex clinical conditions that can nega- not only because patients are largely asymptomatic,
tively affect reproductive potential. In this chapter, but also because some of these infections may actually
we discuss sites of genitourinary tract infections, infec- represent contamination from commensal, nonpatho-
tious organisms, the numerous ways in which leuko- genic organisms. Additionally, due to antibacterial
cytes may impair male reproduction, and the diagnosis effects of semen, there are limitations in culturing all
and treatment of leukocytospermia and bacteriosper- potential pathogenic organisms [1,5]. Several investi-
mia. For the purpose of this chapter, leukocytospermia gators have demonstrated that bacterial isolates from
refers to the World Health Organization threshold of ≥ semen of men in subfertile couples are often commen-
1 × 106 white blood cells/mL in the semen. sal, nonpathogenic organisms. Furthermore, these
isolates often do not correlate with leukocytospermia
Genital tract infections (WHO) or pregnancy rates [6,7].
The male genital tract proximal to the urethra is typi-
cally sterile, and several components of the male repro-
Sites of infection
ductive system are immunologically sequestered [1,2]. Urethritis
Without a fully competent immune response, the Infections of the urethra are most commonly due to
genital tract may be prone to infections. These infec- sexually transmitted pathogens, and they are sep-
tions may be either bacterial or viral, and manifest in arated into two broad categories: gonococcal urethri-
acute, chronic, or subclinical processes. Infections tis (due to Neisseria gonorrhoeae) and nongonococcal
of the male genitourinary (GU) tract can adversely urethritis (Chlamydia trachomatis, Mycoplasma
impact male fertility by impairing spermatogenesis, species, Trichomonas vaginalis) (Fig. 17.1). Patients
disrupting sperm function, causing obstruction of with urethritis typically present with urethral dis-
the genital ductal system, inhibiting accessory gland charge and dysuria. However, asymptomatic carrier
function, or causing inflammation secondary to leu- states may occur, especially in partners of those with
kocyte response [1,3]. known infections. The proper diagnostic workup of
Acute infections of the genital tract are indicated by suspected urethritis includes: (1) Gram stains of a ure-
patient history and can present with irritative voiding thral smear, (2) leukocyte quantification of a first-void
symptoms, urethral discharge, orchialgia, or painful urine sample, and (3) specific isolation techniques such
ejaculation. Traditional diagnostic criteria consist of as endourethral culture, PCR, or ELISA [8]. Based on
two or more of the following parameters: (1) history these modalities, a urethral Gram stain containing > 4
of GU infection and/or abnormal rectal examination, leukocytes per microscopic field (×1000) or a first-
(2) abnormal expressed prostatic secretions and/or void urine sample smear containing > 15 leukocytes
urinary sediment after prostatic massage, (3) ≥ 103 per microscopic field (×400) is regarded as a positive
bacteria/mL ejaculate, or (4) ≥ 106 leukocytes/mL result [9,10]. Consideration should be given to empiric
ejaculate [4]. treatment for both gonococcal and nongonococcal

Infertility in the Male, 4th edition, ed. Larry I. Lipshultz, Stuart S. Howards, and Craig S. Niederberger. Published by Cambridge University
Press. © Cambridge University Press 2009.

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 Chapter 17: Genital tract infection and inflammation

PROSTATITIS URETHRITIS
Escherichia coli Neisseria gonorrhoeae
Klebsiella spp. Chlamydia trachomatis
Proteus spp. Mycoplasma
Pseudomonas aeruginosa Trichomonas vaginalis
Enterococcus spp.

ORCHITIS
Mumps
EPIDIDYMITIS
Coxsackie B
Neisseria gonorrhoeae Neisseria gonorrhoeae
Chlamydia trachomatis Chlamydia trachomatis
Escherichia coli Escherichia coli
Klebsiella spp.
Pseudomonas aeruginosa
Staphylococcus spp.
Streptococcus spp.

Fig. 17.1. Sites of infection and common pathogens in the male genitourinary tract.

organisms when a diagnosis of urethritis is suspected, Patients usually present with constitutional symptoms,
as co-infection is common and the responsible patho- perineal or prostatic pain, and voiding complaints [14].
gens are often unknown at the time of initial evalua- Complications include prostatic abscess, especially in
tion. Typically, a one-time dose of ceftriaxone and a immunocompromised patients and diabetics. A long-
week of doxycycline therapy are recommended [11] or term course of antibiotics for 2–3 weeks is the mainstay
one 2 g dose of azithromycin. of treatment for uncomplicated cases. Type II prosta-
titis is defined as prostatic inflammation and recur-
Prostatitis rent UTI with a bacterial pathogen localized to the
Drach et al. described the first widely used classification prostate. Common pathogens include E. coli and other
system for prostatitis. Briefly, the authors presented UTI-causing Gram-negative organisms (Klebsiella
four classes based on localization techniques and clin- spp., Proteus spp., P. aeruginosa, Enterococcus spp.) [15]
ical correlates: acute bacterial prostatitis (ABP), chronic (Fig. 17.1). Unlike type I prostatitis, type II prostatitis
bacterial prostatitis (CBP), nonbacterial prostatitis is typically not associated with fever. However, both
(NBP), and prostatodynia (Pd) [12]. This classification type I and type II prostatitis are often alleviated with
is no longer commonly employed, and the National antibiotics. Treatment requires extended therapy (as
Institutes of Health (NIH) system is now preferred. The long as 6–12 weeks) with antibiotics that are lipophilic,
NIH system categorizes prostatitis into four main thus able to penetrate the prostate. Trimethoprim-
divisions: type I – acute bacterial, type II – chronic sulfamethoxazole and fluoroquinolones [15] are exam-
bacterial, type III – chronic (abacterial) prostatitis/ ples of antibiotics usually effective in this setting. Care
chronic pelvic pain syndrome (CP/CPPS), and type must be taken in administering prolonged courses
IV – asymptomatic inflammatory [13]. Type I pros- of fluoroquinolones, because of the increased risk of
296 tatitis is generally the result of ascending urinary tract associated tendonitis and tendon rupture [16]. Type III
infection (UTI) of typical uropathogenic organisms. prostatitis or CP/CPPS is a complex entity consisting

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 Chapter 17: Genital tract infection and inflammation

of prostatic pain and voiding difficulties, with 90% of (Fig. 17.1). Paramyxovirus, which causes mumps,
symptomatic prostatitis patients grouped into this cat- is one viral agent capable of inducing significant GU
egory [13]. Type III prostatitis is further divided into pathology. Mumps infrequently involves the testi-
type IIIA and type IIIB. Type IIIA, or inflammatory cles in prepubertal boys, though orchitis occurs in
type, presents with leukocytosis on expressed pros- approximately 20% of affected postpubertal males
tatic secretions (EPS) or post-prostatic massage uri- [22]. Testicular pain and enlargement typically occur
nalysis. Type IIIB, or noninflammatory, is also known 4–6 days after the onset of parotitis, but testicular
as Pd. Interestingly, Nickels et al. demonstrated that a involvement is subclinical in 30–40% of cases. Orchitis
12-week course of antimicrobial therapy does allevi- is bilateral in approximately 30% of affected patients.
ate symptoms in CP/CPPS [17], despite the absence of Historical data reveal associated infertility rates of 25%
documented bacterial infection that defines this condi- in bilateral disease, but this problem is uncommon
tion. Lastly, type IV prostatitis is an incidental finding today due to the advent of the mumps vaccine [22,23].
on prostate biopsy or expressed prostatic secretions. Testicular atrophy is observed in 30–50% of patients
The effect of prostatitis on male fertility will be dis- with mumps orchitis, and infertility may be an associ-
cussed later in this chapter. ated long-term consequence [19]. Like paramyxovirus,
coxsackie B virus may also result in viral orchitis simi-
Epididymitis lar to mumps [22].
Epididymitis is one of the most common causes of acute Bacterial orchitis, unlike viral orchitis, often results
scrotum, presenting with ipsilateral scrotal swelling from contiguous spread of infection from the epididy-
and pain. Systemic symptoms such as fever and leuko- mis. Common pathogens include N. gonorrhoeae/C. tra-
cytosis are seen in 30–50% of patients [18]. The organ- chomatis, Gram-negative bacilli (E. coli, K. pneumoniae,
ism responsible for the infection typically is dependent P. aeruginosa), and Gram-positive cocci (staphylococci
on patient demographics. Sexually active men under and streptococci) (Fig. 17.1). Patients may present with
the age of 35 are often infected with C. trachomatis fever, testicular pain and swelling, and often an acute,
or N. gonorrhoeae, with accompanying symptoms reactive hydrocele [22]. Medical management is simi-
of urethritis (Fig. 17.1). In many patients, exposure lar to that administered in the case of epididymitis, as
to the organism may have occurred several months treatment is pathogen-specific with consideration to
before presentation [10]. Chlamydia accounts for two- the given patient demographic. Surgical intervention
thirds of the total cases of urethritis in these men and is indicated for testicular infarction or abscess [22].
is two to three times more common than gonorrhea
[19]. Similar to urethritis, co-infection is not uncom- Specific bacterial infections of the male
mon in epididymitis, occurring in approximately 30%
of cases [19]. For men over 35 years of age, men with genital tract
recent urinary tract instrumentation or surgery, or Neisseria gonorrhoeae
men with anatomical abnormalities, E. coli is often N. gonorrhoeae is a common cause of urethritis in
the etiological organism causing epididymitis [10]. America [24]. Although most affected males are symp-
Treatment of epididymitis should be pathogen-spe- tomatic, as many as 10% of men with N. gonorrhoeae
cific, with consideration given to patient demographics urethritis are asymptomatic [25]. Ascending infec-
during initial evaluation. tions in men involving genitourinary tract structures
In the case of acute epididymitis, several authors other than the urethra are rare (estimated at less than
have observed that both sperm concentration and 1%), but when epididymitis or orchitis does occur,
forward motility are transiently impaired [20,21]. testicular damage or excurrent ductal obstruction
However, with appropriate antibiotic treatment, can result. In a study of 184 men seen at an infertility
decreased semen parameters and impaired sperm clinic, patients with a prior remote history of N. gonor-
function often normalize [21]. rhoeae had significantly more seminal leukocytes than
those without such a history (2 × 106/mL vs. 0.5 × 106,
Orchitis P < 0.0013) [26].
Viruses represent a significant reproductive tract
pathogen, capable of causing testicular inflamma- Chlamydia trachomatis
tion and infection. These infections typically arise via C. trachomatis is the most common cause of non- 297
hematogenous routes rather than by contiguous spread gonococcal urethritis and acute epididymitis in men

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 Chapter 17: Genital tract infection and inflammation

of reproductive age, and is also the most common infertile couples diagnosed with chlamydia using LCR
sexually transmitted disease in industrial nations compared to normal controls [48]. In a study of men
[27]. Ten to twenty-five percent of infected men may be undergoing an infertility evaluation, Hosseinzadeh
asymptomatic with chronic, subclinical disease [28]. et al. also noted that semen presenting as PCR-positive
Infertility may arise because C. trachomatis is an obli- for chlamydia was not associated with impairment of
gate intracellular organism capable of causing a variety sperm motility [49]. However, the authors did report
of pathological changes including epididymitis, orchi- a twofold increased incidence of leukocytospermia in
tis, testicular atrophy, genital ductal obstruction, ger- PCR positive semen samples.
minal epithelial cell damage, and blood–testis barrier
breakdown leading to antisperm antibody production Mycoplasma
[1,29]. This bacterium exists in two forms – the reticu- Four species of mycoplasma exist, two of which are
late body (RB) and the elementary body (EB). As an RB, pathogenic in human beings: Mycoplasma hom-
C. trachomatis actively replicates in host cells, while the inis and Ureaplasma urealyticum. U. urealyticum is
inactive EB serves as an extracellular agent of transmis- a significant cause of urethritis, and is believed to be
sion. Immunologic laboratory techniques for diagno- responsible for up to 25% of nongonococcal urethri-
sis are targeted against EB antigens [1]. Hosseinzadeh tis [1,5]. Similar to other bacterial causes of urethritis,
et al. demonstrated that EBs cause increased tyrosine mycoplasma infections may present acutely or remain
phosphorylation of sperm proteins, possibly impairing subclinical, and the actual impact of U. urealyti-
sperm capacitation and function [30]. Interestingly, cum and M. hominis on male fertility is controversial
subsequent data revealed that co-incubation of human (Tables 17.2, 17.3) [53–67].
spermatozoa and C. trachomatis EBs causes decreased Gnarpe and Friberg initially demonstrated that,
motility and increased premature sperm death [31]. although U. urealyticum is present in both fertile and
The spermicidal effects of EBs are mediated specifically infertile couples, it is more prevalent in the latter
by lipopolysaccharide (LPS), ultimately resulting in [53]. Furthermore, Swenson et al. found that infer-
apoptosis [32,33]. In sum, EBs represent an important tile males with U. urealyticum infections had signifi-
mediator of C. trachomatis-induced pathophysiology. cantly improved sperm motility following treatment
The relationship between C. trachomatis infec- [66]. Additionally, Toth et al. demonstrated that cou-
tion and semen quality is an actively debated topic ples in whom the male’s U. urealyticum infection was
(Table 17.1) [34–49]. Multiple studies have demon- cleared with doxycycline experienced significantly
strated a detrimental impact on semen parameters higher pregnancy rates than couples with persistent
[34–36] and stimulation of antisperm antibody (ASA) male infection [67]. However, sperm count and overall
production [37,38]. However, other investigators semen quality were not significantly different between
have found no relationship between chlamydial infec- the two groups.
tions and semen parameters, ASA production, sperm Other authors have noted that men with U. urea-
penetration, or leukocytospermia [39,40–47]. Several lyticum infections have reduced sperm count with
authors suggest these discrepancies may be explained reduced concentration, motility, and morphology [54–
by the lack of uniformity and reliability in the diagnos- 56]. Rose and Scott demonstrated that semen samples
tic methods employed in these various studies, i.e., cul- with mycoplasma (U. urealyticum and M. hominis)
tures (ejaculates and urethral swabs), enzyme-linked had decreased motility, morphology, hyperactivation,
immunoassays to chlamydial antigens, and serum and and acrosome reactivity when compared with controls
semen antibodies [29,49]. Efforts to diagnose chla- [68]. A more recent in-vitro study also noted decreased
mydial infections are further hampered by the fact motility and increased membrane alterations on the
that seminal plasma inhibits cell culture techniques basis of the hypo-osmotic swelling test [69]. Lastly,
[50] and the host antibody response is not specific to Reichart et al. demonstrated that U. urealyticum is
C. trachomatis [51]. More recently, molecular amp- associated with increased sperm chromatin and DNA
lification techniques consisting of polymerase chain damage on the basis of both in-vitro studies (semen
reaction (PCR) and ligase chain reaction (LCR) have from normospermic men inoculated with U. urealyti-
come to represent the tests of choice for diagnosing cum) and in-vivo studies (semen culture positive for
chlamydial infections [52]. Eggert-Kruse et al. dem- U. urealyticum) [70]. The authors hypothesized that
298 onstrated no effect on semen parameters in men from these changes may ultimately affect embryogenesis. In

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 Chapter 17: Genital tract infection and inflammation

Table 17.1. Studies investigating chlamydial infections in asymptomatic males attending infertility clinics
Reference n Detection method Prevalence Findings
Studies supporting a role for chlamydia
Custo, 1989 [34] 1023 EIA 9% Men with C. trachomatis infection had a significantly
higher incidence of abnormal “semen scores” than
healthy controls + symptomatic men infected with
common pathogens + symptomatic men without
infection
Wolff, 1991 [35] 209 EIA for seminal 15.4% Reduced ejaculate volume and progressive sperm
prevalence antibodies motility in C. trachomatis antibody-positive patients
analysis with higher seminal inflammation (> 250 ng PMN
331 semen elastase/mL) than those with lower inflammation
Analysis (< 250 ng PMN elastase/mL)
Cengiz, 1997 [36] 284 EIA for seminal 12.6% Significant differences in density, morphology,
antibodies motility, and viability between antibody-positive and
antibody-negative patients
Witkin, 1995 [37] 227 EIA for seminal and IgA 24.7% Men with antichlamydial IgA in their semen had a
serum IgA and IgG (semen), lower median sperm count than those without ASA
antibodies 14.5% (serum) strongly correlated to antichlamydial IgA in semen
IgG 10.9%
(semen),
21.5% (serum)
Munoz, 1995 [38] 48 EIA for seminal and IgA 29.2% Men with seminal antichlyamdial IgA had elevated
serum IgA and IgG (semen) IgG seminal concentrations of specific T-cell population
antibodies 8.3% (semen) than men without
No differences in semen parameters in patients with
antichlamydial antibodies (IgA and/or IgG) than men
without
Studies refuting a role for chlamydia
Close, 1987 [39] 270 Micro-IF of serum 5.9% No difference in sperm count, motility, forward
progress, % ovals, number of leukocytes, SPA, or serum
ASA between patients seropostive and seronegative
for C. trachomatis
Nagy, 1989 [40] 184 McCoy culture with 14.1% No difference in cell count, motility, morphology,
Lugol stain bovine mucus penetration, and HOS in infected and
noninfected groups
Rujis, 1990 [41] 184 Direct IF of urethral Smears 2.8% No association between chlamydial antibody titers
smear, serum and and sperm count, motility, morphology, leukocytes,
seminal antibody MAR, SCMC results
titers
Soffer, 1990 [42] 175 McCoy culture and 7% No differences in mean motility index, total sperm
Iodine stain count, normal form percentage between infected and
noninfected groups
Seminal ASA activity was elevated in cases of positive
culture (both C. trachomatis and mycoplasma)
Bjercke, 1992 [43] 100 EIA for seminal and IgA 24% No difference in sperm concentration, total sperm
serum IgA and serum (semen) count, motility (several parameters), and morphology
IgG serum IgA/IgG
not reported
Dieterle, 1995 [44] 50 C. trachomatis PCR 10% No association between the detection of C.
and direct DNA trachomatis (PCR or DNA probing) and chlamydial
probing antibodies in serum or semen
No association between chlamydial antibodies in
serum or semen and mean sperm concentration,
progressive motility, morphology, and serum ASA
Eggert-Kruse, 197 EIA of semen (both IgA 18.8% IgG No association between seminal chlamydial
1996 [45] IgA and IgG) 8.1% antibodies and sperm count, motility, morphology, 299
viability, MAR, SCMPT, and PCT

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 Chapter 17: Genital tract infection and inflammation

Table 17.1. continued

Reference n Detection method Prevalence Findings
Eggert-Kruse, 1317 Indirect 12.6% No association between seminal chlamydial
1997 [46] immunofluorescence antibodies and sperm count, motility, morphology,
assay (IFA) MAR, SCMPT, and PCT
Habermann, 1999 207 EIA of semen IgA 18.8% IgG No difference in sperm parameters, leukocytes,
[47] 15.5% and MAR in patients with and without chlamydial
antibodies
Eggert-Kruse, 707 LCR of semen and 1.8% No association between chlamydial infection by LCR
2003 [48] urine samples and sperm count, motility, morphology, viability, MAR,
and SCMPT
Hosseinzadeh, 642 LCR/PCR of semen 4.9% Men whose ejaculates were PCR-positive for
2004 [49] samples chlamydial DNA had a higher mean concentration
of leukocytes and twice the prevalence of
leukocytospermia compared with those whose
ejaculates were PCR-negative No difference in
sperm concentration, % motile sperm, or % normal
morphology in men PCR-positive and men PCR-
negative for chlamydia
ASA, antisperm antibody; DNA, deoxyribonucleic acid; EIA, enzyme immunoassay; HOS, hypo-osmotic swelling; IF,
immunofluorescence; Ig, immunoglobulin (IgG typically found in serum, IgA typically found in secretions); LCR, ligase chain reaction;
MAR, mixed agglutination reaction; PCR, polymerized chain reaction; PCT, postcoital testing; PMN, polymorphonuclear leukocyte;
SCMC, sperm–cervical mucus contact; SCMP, sperm cervical mucus penetration test.

Table 17.2. Studies investigating mycoplasma infections in males attending infertility clinics
Reference n Detection Prevalence Findings
method
Studies supporting a role for mycoplasma
Gnarpe, 1972 [53] A: 36 infertile Culturea Patient groups UU was isolated in higher frequencies in the couples
couples B: 19 A: 85% with infertility (A: primary infertility; B: ASA detected
infertile couples B: 95% Controls in female partners) than in the two control groups
C: 23% (C: pregnant women; D: men married to pregnant
D: 26% women)
Fowlkes, 1975 625 male partners Culture 39% Patients with UU infections had changes in their
[54] of infertile couples distribution curves toward lower total sperm counts
with > 106/mL and lower motility than infertile patients free of UU
semen infections
Patients with UU infections had a significant increase
in the aberrant sperm forms compared with infertile
patients free of UU infections
Upadhyaya, 1984 280 attending a Culture 50% Patient with UU infections had significantly lower
[55] fertility clinic sperm concentrations
UU infection did not affect sperm motility, viability,
or morphology
Naessens, 1986 120 men Culture 33% Isolation of UU was significantly correlated with
[56] attending an lower sperm counts and abnormal motility, but was
infertility clinic not correlated with sperm morphology
Kalugdan, 1996 34 males PCR–ELISA 29.4% Mycoplasma-positive spermb demonstrated
[57] decreased oocyte penetration capacity compared
with mycoplasma-negative sperm
Studies refuting a role for mycoplasma
De Louvois, 1974 120 infertile Culture M. hominis Fertile Mycoplasma was not more prevalent in infertile
[58] couples 13.2% versus fertile couples
Infertile 14.7% U.
300 urealyticum
Fertile 52.6%
Infertile 57.2
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 Chapter 17: Genital tract infection and inflammation

Table 17.2. continued

Reference n Detection Prevalence Findings
method
Harrison, 1975 120 infertile Culture M. hominis Mycoplasma was not more prevalent in infertile
[59] couples; 27 Conception(+): 18% couples who conceived versus those who did not
couples conceived Conception(–): 18% conceive
during the 1 year U. urealyticum
of study Conception(+): 63%
Conception(–): 56%
Desai, 1980 [60] 150 infertile Culture Couples: 46% No statistical difference in sperm count, motility, and
couples Men: 32.6% morphology between infertile patients with and
without UU infections
Cintron, 1981 96 male partners Culture 45% No statistical difference in sperm count, motility, and
[61] of infertile couples morphology between infertile patients with and
without UU infections
Lewis, 1981 [62] 31 men attending Culture 58% No statistical difference in the distribution of
an infertility clinic “normal” and “abnormal” semen analyses between
infertile patients with and without UU infections
Shalhoub, 1986 76 male partners Culture Fertile: 27% No statistical difference in sperm count, motility,
[63] of infertile couples Infertile: 21% morphology, BCMP, and SPA between infertile
and 22 fertile patients with and without UU infections
controls
de Jong, 1990 569 infertile men Culture Fertile: 5.3% UU was not more prevalent in infertile versus fertile
[64] 75 fertile controls Infertile: 7% couples
No statistical difference in sperm count, motility,
and morphology between infertile men with and
without UU
Ombelet, 1997 143 infertile Culture M. hominis No further investigations of mycoplasma infection
[65] couples Fertile: 1.4% were performed
144 fertile couples Infertile 2.9% U.
urealyticum
Fertile: 7.6%
Infertile: 12.5%
ASA, antisperm antibodies; BCMP, Bovine cervical mucous penetration; ELISA, enzyme-linked immunosorbent assay; PCR,
polymerized chain reaction; SPA, hamster sperm penetration assay; UU, U. urealyticum.
a
Various semen culture methods were employed by the different studies.
b
Processed by the 48 hr TEST (TES and Tris) yolk buffer method.

Table 17.3. Studies investigating treatment of mycoplasma infections in males attending infertility clinics
Reference n Detection Treatment course Findings
method
Swenson, 73 men with Culture Both partners: tetracycline The 64 patients successfully treated for UU had a
1979 [66] infection 250mg QID or doxycycline significant improvement in sperm motility (speed
attending an 100mg BID for 30 days of forward progression and % motility)
infertility clinic In the 64 patients successfully treated for UU, the
median percentage of motile cells increased from
35% to 50% following treatment
Toth, 1983 161 men with Culture Both partners: doxycycline In couples treated for UU infection, eradication
[67] infection 100mg BID for 28 days of UU in the male partner resulted in significantly
attending an more pregnancies than in couples whose male
infertility clinic partner did not clear the infection (60% vs. 5%)
Harrison, 1975 88 couples with Culture 30 couples: doxycycline In couples treated for mycoplasma infection,
[59] primary infertility 100mg BID doxycycline therapy was shown to clear
28 couples: placebo infection (96% eradication rate) but did not affect
30 couples: no intervention conception rates in those treated versus controls
(28 days for all) 301
UU, U. urealyticum.

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 Chapter 17: Genital tract infection and inflammation

sum, mycoplasma may lead to infertility by impairing male-factor fertility is controversial, several stud-
sperm function and genetic integrity. ies suggest that the adverse effects on sperm func-
Several authors have examined the pathological tion arise from ROS production, DNA damage, and
interaction between mycoplasma and sperm. Gnarpe direct attachment of mycoplasma to spermatozoa.
and Friberg observed that U. urealyticum attached dir- Therefore, treating patients with an appropriate
ectly to spermatozoa. This finding has been confirmed course of antimicrobial therapy such as doxycy-
by others using electron micrography, noting specific cline should be considered, as doxycycline eradicates
attachment to the sperm head and midpiece regions infection in the majority of cases [58,59,64,67].
[69,71,72]. As a result of this direct interaction, sperm In regard to in-vitro fertilization (IVF), current
motility may be impaired. Furthermore, Busolo and data suggest that adverse outcomes (decreased preg-
Zanchetta observed decreased hamster egg penetration nancy rates) associated with U. urealyticum infection in
in vitro (using viable sperm cultured with U. urealyti- couples are secondary to endometritis associated with
cum) [73]. Kalugdan et al. corroborated these results by U. urealyticum infection [75,76]. As a result, treatment
comparing penetration of zona-free hamster oocytes of male partners with U. urealyticum infection prior to
using “mycoplasma-positive sperm” and “mycoplas- IVF is still recommended [77].
ma-negative sperm.” Mycoplasma positivity or nega- The methods employed to detect U. urealyticum
tivity was determined by extracting sperm DNA from have evolved over time. Classically, genital tract sam-
washed semen samples and assaying for mycoplasma ples were inoculated in broth with an indicator sys-
DNA using PCR [57]. These last two studies suggest tem utilizing the presence of urease in U. urealyticum.
that direct attachment of mycoplasma to sperm may As urea is metabolized into ammonia and ultimately
impair sperm penetration of eggs. Interestingly, Potts ammonium, the pH of the medium is increased, result-
et al. demonstrated that semen samples from men pre- ing in a color change of the indicator system. This color
senting for a chronic prostatitis evaluation who were change corresponds to adequate bacterial replication.
culture-positive for U. urealyticum had significantly At this point, the broth is subcultured on selective solid
elevated reactive oxygen species (ROS) compared media for final identification. In recent years, PCR tech-
with those who were culture-negative [74]. Therefore, niques (targeted to the urease gene) have offered faster
ROS may also lead to damaged sperm membranes and alternatives to culture for the diagnosis of mycoplasma.
DNA, providing additional mechanisms for fertility Blanchard et al. reported a 97.3% sensitivity and a 72%
impairment. specificity of PCR compared to culture (considered the
In contrast to the above findings, de Louvois et al. “gold standard”) in 250 genital swabs from 181 male
did not observe an increased incidence of U. urealyti- and female patients [78]. Furthermore, Teng et al.
cum or M. hominis in infertile versus fertile couples reported a higher sensitivity for U. urealyticum detec-
[58]. Harrison et al. noted that in 120 couples present- tion with PCR (semen, urine, urethral swabs, or pros-
ing for infertility evaluation, infection with U. urealyti- tatic secretions) than culture in males with urethritis
cum or M. hominis did not affect the rate of conception or infertility [79]. However, Povlsen et al. reported only
during 12 months of follow-up [59]. Additionally, in a a 64% sensitivity and a 99% specificity for U. urealyti-
subgroup of 88 patients with primary infertility, doxy- cum detection with PCR compared to culture in male
cycline therapy was shown to clear infection but did urogenital swabs [80]. Of note, Blanchard states that
not affect conception rates. More recently, de Jong et patient samples may contain substances that inhibit
al. and Ombelet et al. noted no difference between the PCR, as suggested by increased PCR sensitivity with
frequency of U. urealyticum in the semen of infertile dilution of specimens. This finding may account for the
and fertile men [64,65]. Furthermore, several studies decreased sensitivity of PCR versus culture. Further
demonstrated no statistical difference in sperm count, studies employing PCR for U. urealyticum detection in
motility, or morphology when infertile patients with infertile males are needed to help clarify this issue.
and without U. urealyticum infections were compared
[60–62]. These results were corroborated by Shalhoub Gram-negative infections
et al., who found no significant differences in semen Gram-negative infections, in particular E. coli, are
parameters, mucus penetration, or oocyte penetration the most common cause of nongonococcal bacte-
between semen samples with and without U. urealyti- rial infections of the male genital tract. While these
302 cum [63]. Although the impact of mycoplasma on infections are often symptomatic, they may manifest

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 Chapter 17: Genital tract infection and inflammation

as subclinical epididymitis or prostatitis [1,2,81]. In Overall, the incidence of Gram-negative infec-

aggregate, the numerous clinical studies examining the tions is low in young men [1,3]. While several in-
effects of E. coli on male fertility do not support det- vitro studies suggest mechanisms for male fertility
rimental associations. Fowler and Mariano could not impairment by Gram-negative organisms, a paucity
detect E. coli in any ejaculates of 62 male partners of of similar findings in the clinical literature suggests
infertile couples using IgA antibodies targeted against that these organisms may have limited male repro-
eight common O-antigens [82]. Hiller et al. isolated ductive pathogenicity.
E. coli by culture in less than 10% of semen samples
from 37 males attending an infertility clinic; however, Trichomonas vaginalis
no association with sperm dysfunction or leukocyto- T. vaginalis is a sexually transmitted protozoan organ-
spermia was observed [83]. Naessens et al. noted ism that accounts for a minority of nongonococcal ure-
similar results in 120 male infertility patients [56]. thritis cases [1]. Tuttle et al. demonstrated that, in an
Furthermore, Ombelet et al. observed no difference in-vitro environment, sperm incubated with T. vagina-
in the prevalence of E. coli when comparing 144 fertile lis had diminished motility that significantly decreased
males with 143 subfertile males [65]. More recently, over time [90]. In subsequent in-vitro studies, Jarecki-
Esfandiari et al. found no correlation between Gram- Black et al. isolated a spermicidal protein released by
negative bacteria (Enterobacteriaceae family) and T. vaginalis [91]. Furthermore, Gopalkrishnan et al.
sperm characteristics in 80 infertile men with asymp- reported that T. vaginalis-positive semen from asymp-
tomatic leukocytospermia [84]. These studies collec- tomatic men (4.4% of cases) demonstrated decreased
tively suggest that the presence of E. coli in the semen of sperm motility, decreased morphology, altered mem-
infertile males often represents benign colonization or brane integrity, and increased viscosity versus con-
sample contamination rather than an infectious cause trols. Treatment with a single course of metronidazole
of infertility. resulted in significantly improved semen parameters in
Despite the lack of a definitive association between 50% of patients [92]. Interestingly, Lloyd et al. recently
E. coli and male fertility impairment from the clinical described the first case of T. vaginalis orchitis associ-
literature, several basic science studies demonstrate ated with oligoasthenoteratozoospermia and hypogo-
that E. coli binds to sperm and causes ultrastructural nadism diagnosed during testicular sperm extraction
damage and sperm agglutination. Collectively, these [93]. These limited data suggest that T. vaginalis may
changes result in decreased sperm motility and pene- impair sperm function, though the clinical utility of
tration. Teague et al. first reported that E. coli obtained screening for T. vaginalis remains to be seen.
from urinary and cervical cultures, when added to
semen from healthy volunteers, resulted in agglutin- Viruses
ation of spermatozoa and decreased motility [85]. These Viruses may impact male fertility by stimulating
effects were found to be independent of endotoxin or direct cytotoxic immune mechanisms and indirect
lipopolysaccharide (LPS). Wolff et al. incubated sperm inflammatory processes (Table 17.4) [94–102]. They
from healthy donors with E. coli isolated from the can infect the genitourinary system via the blood stream
ejaculates of male infertility patients. They observed or, less commonly, via the urethra. Viruses depend on
sperm agglutination due to direct adherence of E. coli host cells for replication, and these pathogens are cap-
to sperm mediated by mannose-binding proteins [86]. able of invading epithelial, neuronal, and immune cells
Interestingly, reversal of mannose-mediated agglutin- present in the genitourinary tract [3]. Viruses are effect-
ation yielded return of sperm motility. El-Mulla et al. ively detected in semen by sensitive and specific DNA
demonstrated decreased inducibility of the acrosome amplification techniques including PCR and LCR.
reaction due to E. coli in an in-vitro experiment [87]. The herpesvirus family consists of herpes simplex
Studies by Diemer et al. suggest that at prolonged virus (HSV) types 1 and 2, cytomegalovirus (CMV),
incubation times E. coli exerts irreversible damage to Epstein–Barr virus (EBV), varicella-zoster virus
both acrosomal and tail architecture [88,89]. Though (VZV), and human herpes virus (HHV) types 6, 7,
these results suggest a role for E. coli in male infertil- and 8. These viruses are collectively responsible for an
ity, high concentrations of E. coli were used (1 : 1 ratio array of disease processes ranging from simple rashes
with sperm), and therefore they may not be clinically to malignancy. Krause et al. evaluated the presence of
relevant [89]. HSV, CMV, and EBV in the semen of infertile men with 303

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 Chapter 17: Genital tract infection and inflammation

Table 17.4. Studies investigating viral infections in asymptomatic males attending infertility clinics
Reference Virus(es) Detection Study Prevalence Findings
method population (n)
Krause, 2002 HSV, CMV, Serum IgG and Male infertility (+) Of all the viruses and antibody
[94] EBV IgM serology patients with Leukocytospermia: isotypes, only IgM against HSV
(130) and without HSV IgM: 10% was more prevalent in patients
leukocytospermia (–) with leukocytospermia than
(80) Leukocytospermia: those without
HSV IgM: 1.25%
Kapranos, HSV, CMV, DNA PCR Men attending an HSV: 49.5% HSV, but not CMV or EBV, was
2003 [95] EBV infertility clinic (113) CMV: 7.1% associated with decreased mean
EBV: 16.8% sperm count and mean sperm
motility
Shen, 1994 CMV DNA PCR Male partners of 32.7% Associations with semen
[96] infertile couples parameters or sperm function
(217) were not studied
Yang, 1995 CMV CMV IgG serum Infertile couples IgG: 98.9% In males, no effects on semen
[97] serology and (250) DNA PCR: 33.5% quality were observed
DNA PCR of
semen samples
Levy, 1997 CMV CMV IgG serum Male partners of IgG: 58% Authors recommend screening
[98] serology and infertile couples (81) DNA PCR: 2.85% for CMV-seropositive patients
DNA PCR of tested for serology followed by viral PCR detection
semen samples and 70 separate in semen due to potential risk of
semen samples congenital CMV infection
from CMV IgG
positive patients for
PCR analysis
Bezold, 2002 Herpesvirus DNA PCR Men seeking fertility All types: 17.1% Patients with EBV-positive PCR
[99] types: evaluation (252) EBV: 7.1% had higher seminal granulocytes
EBV, HSV, HSV: 3.2% than those without
CMV, HHV CMV: 3.6% No associations between any
6–8, VZV HHV 6: 4% virus and sperm count, motility,
HHV 7: 0.4% and morphology Authors also
HHV 8: 0% recommend screening for HSV
VZV: 0% and CMV due to potential risk of
congenital infections
Green, 1991 HPV 11 & 16 DNA PCR Men attending an STD clinic: 85% Associations with semen
[100] STD clinic (27) and a Fertility clinic: parameters or sperm function
fertility clinic (104) HPV total: 41% were not studied
HPV 11: 22%
HPV 16: 33%
HPV 11&16: 14%
Lai, 1997 HPV 16 & 18 DNA & RNA Men attending a HPV 16 Higher rate of asthenospermia
[101] PCR fertility clinic (24) DNA: 25% in patients infected with either
RNA: 8% HPV than in patients without
HPV 18 infection Certain aspects of
DNA: 46% motility were significantly lower
RNA: 8% in the specimens with HPV than
in those without
Martorell, HPV DNA PCR Patients with 6.48% Further studies indicate that
2005 [102] (multiple from paraffin azoospermia Sertoli, Leydig, and possibly
types) embedded undergoing germinal cells harbored the HPV
sections testicular biopsy infections
(185)
CMV, cytomegalovirus; DNA, deoxyribonucleic acid; EBV, Epstein–Barr virus; HHV, human herpes virus; HPV, human papillomavirus;
HSV, herpes simplex virus; Ig, immunoglobulin (IgG typically found in serum, IgA typically found in secretions); PCR, polymerized chain
reaction; STD, sexually transmitted disease; VZV, varicella-zoster virus.
304

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 Chapter 17: Genital tract infection and inflammation

and without leukocytospermia [94]. Through the use clinic, Lai et al. demonstrated that patients infected
of serum serologies, the authors noted significantly with HPV type 16 or 18 detected by PCR in semen
more patients with serum IgM antibodies against HSV had a significantly higher rate of asthenospermia
when comparing infertile men with leukocytospermia than patients without HPV infection (75% vs. 8%,
with controls (10.8 vs. 1.25, P < 0.05). However, when P = 0.008) [101]. No such differences were observed
they compared HSV DNA PCR on semen samples from regarding sperm concentration or morphology when
infertile men with leukocytospermia to semen samples comparing patients with and without seminal HPV.
from infertile men without leukocytospermia, they More recently, Martorell et al. isolated HPV by PCR
found no such association. More recently, Kapranos in 6.48% of 185 paraffin-embedded testicular biopsies
et al. employed PCR detection of HSV virus in the of men with nonobstructive azoospermia versus 0%
semen of 113 men attending an infertility clinic [95]. of healthy controls (autopsies) [102]. However, each
The investigators detected HSV in 49.5% of samples of the 12 patients identified with HPV infection had
and reported significantly decreased sperm concentra- other probable etiologies for azoospermia (e.g., chem-
tion and motility in HSV-positive patients versus HSV- otherapy effects, etc.). At this time, the relationship
negative patients. CMV and EBV were also studied, but between HPV and male infertility is unclear, given the
neither impacted semen parameters. available data.
Yang et al. studied the presence of CMV in 250 Infection with human immunodeficiency virus
infertile couples in Taiwan using blood serologies and (HIV) is a significant cause of morbidity and mortal-
DNA hybridization techniques. While the authors ity worldwide, with disease progression resulting in
demonstrated 98.9% prevalence of anti-CMV IgG in acquired immunodeficiency syndrome (AIDS). The iso-
the serum from males, they detected viral shedding in lation of HIV in human semen is well documented [3],
only 33.5% of semen samples using DNA hybridization and thus represents a potential pathogen to sperm devel-
techniques [97]. The authors reported that viral shed- opment and function (Table 17.5) [103–110]. Krieger
ding of CMV in semen did not affect semen quality. In a et al. demonstrated that semen specimens from 21 men
similar study of 81 infertile couples in France, Levy et al. seropositive for HIV did not significantly differ from
noted the presence of anti-CMV IgG serologies in 58% those of seronegative semen donors [103]. However,
of men and 62% of women [98]. However, in 70 semen the authors demonstrated that the three study patients
samples of those with anti-CMV IgG positive serolo- with AIDS had leukocytospermia and microscopically
gies, only two semen samples were positive for CMV abnormal sperm forms. Several subsequent investiga-
DNA through amplification. The difference in these tions corroborate these data, as early HIV infections
two studies is largely attributed to patient demograph- are generally not associated with abnormalities in
ics (France vs. Taiwan), and the full effects of CMV on semen parameters. However, with disease progres-
reproductive health remain to be determined. sion to AIDS (i.e., CD4+ count < 200 cells/mm3 or
In a study by Bezold et al., herpesviruses were symptomatic disease), significant seminal abnormali-
detected in 17% of 252 semen samples from men ties (impaired concentration, morphology, motility,
undergoing an infertility evaluation [99]. EBV was viscosity) and leukocytospermia are often evident
the most common (7.1% of samples), and was associ- [104–107,111]. Using regression analysis, Dondero et al.
ated with increased concentrations of granulocytes. demonstrated correlations between peripheral blood
However, no associations between EBV infection and CD4+ lymphocyte count, sperm motility (r = 0.45,
impaired semen parameters or sexual gland dysfunc- P < 0.05), and abnormal sperm morphology (r = –0.51,
tion (e.g., fructose assay for seminal vesicles) were P < 0.05) in 21 HIV seropositive males [107].
demonstrated. Treatment of HIV with the antiretroviral zidovu-
The relationship of human papillomavirus (HPV) dine (AZT) is not associated with detrimental changes
to male fertility is intriguing, considering the role of in semen parameters [103,104], and in some reports
HPV in genital warts (types 6 and 11) and penile carci- AZT therapy has been shown to result in decreased
noma (types 16 and 18). Through DNA PCR of semen, seminal leukocytes [106] and titers of seminal HIV-1
Green et al. reported a 41% prevalence of seminal [105]. Dulioust et al. studied 189 HIV-infected men
HPV in a total of 104 men attending a fertility clinic without AIDS, a majority (94%) of whom were tak-
[100]. In another study of 24 men attending a fertility ing one or more antiretrovirals [108]. These otherwise
305

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 Chapter 17: Genital tract infection and inflammation

Table 17.5. Studies investigating the effects of HIV, AIDS, and antiretrovirals on male fertility
Reference Study Symptomatic AIDS (n) Patients Findings
population HIV (n) receiving
(n) treatment (%)
Krieger, 1991 24 HIV-positive 5 3 12 (50%) No difference in sperm count,
[103] males morphology, motility, or WBCs in HIV
semen samples compared to controls
Zidovudine treatment did not affect
semen parameters Three patients with
AIDS had grossly visible leukocytospermia,
spermophagy, and abnormal sperm form
by light and electron microscopy
Crittenden, 39 HIV-positive 9 8 9 (49%) HIV-positive males had decreased motile
1992 [104] males sperm, more round cells
Serum CD4 count and CD4 percentage of
HIV patients was positively correlated to
proportion of motile sperm
Anderson, 95 HIV-positive 47 with AIDS or 47 with 31 (33%) 9% of HIV-positive males had positive
1992 [105] males symptoms AIDS or HIV semen cultures Serum CD4 < 200/
symptoms µL, symptomatic disease, and elevated
seminal WBC (>1x106/ml) were associated
with HIV detection in semen
Zidovudine treatment was associated with
decreased detection of HIV in semen
Politch, 1992 166 HIV- NA 55 80 (48%) HIV-positive males with CD4 counts > 200/
[106] positive males µL and patients with zidovudine therapy
(regardless of CD4 count) had normal
semen parameters
Untreated HIV-positive males with CD4
counts < 200/µL had decreased sperm
count and concentration and increased %
of abnormal sperm forms
Dondero, 21 HIV-positive None None 16 (76%) No difference in sperm density, motility,
1996 [107] males and viscosity in HIV patients compared to
controls
HIV patients had a higher number of WBCs
and spermophage cells than controls
Serum CD4 count of HIV patients was
positively correlated to sperm motility and
inversely correlated to abnormal sperm
Dulioust, 189 HIV- None None 177 (94%) HIV-positive males had decreased total
2002 [108] positive males sperm count, decreased % of rapidly
requesting progressive sperm, decreased ejaculated
ART volumes, and increased nonsperm cells
compared with controls
These results may be due to antiretroviral
treatment
Nicopoullos, 106 HIV- None None 74 (55.6%) HIV-positive males had decreased sperm
2004 [109] positive males concentration, total count, progressive
requesting motility, normal morphology, and
ART (IUI) ejaculate volume compared to controls
A significant positive correlation was
observed between CD4 count and sperm
concentration, total count, motility,
progressive motility, and post-preparation
concentrationa A significant negative
correlation was observed between CD4
count with normal sperm morphology of
both raw and post-preparation samples
VL < 1000 copies/mL and the use of
306 antiretrovirals significantly improved IUI
outcomes

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 Chapter 17: Genital tract infection and inflammation

Table 17.5. continued

Reference Study Symptomatic AIDS (n) Patients Findings
population HIV (n) receiving
(n) treatment (%)
Bujan, 2007 190 HIV- None None 172 (91%) HIV-positive males had decreased total
[110] positive males sperm motile count, % progressive
attending a spermatozoa, polynuclear cell count, and
fertility clinic ejaculate volume versus controls. HIV-
positive males had increased seminal pH
value and multiple anomaly indexb versus
controls
These results may be due to antiretroviral
treatment
AIDS, acquired immunodeficiency syndrome; ART, assisted reproductive technology; CD, cluster of differentiation; HIV, human
immunodeficiency virus; IUI, intrauterine insemination; VL, viral load; WBC, white blood cell.
a
Sperm washing of HIV for IUI
b
Mean number of anomalies per abnormal spermatozoon

healthy HIV patients had significant abnormalities viral loads significantly improved the outcome of intra-
in their semen parameters compared to age-matched uterine insemination with sperm washing [109].
seronegative controls, leading the authors to suggest Antiretrovirals may indeed decrease HIV in semen,
a possible association between antiretrovirals and thereby inhibiting HIV-mediated sperm dysfunction.
impaired semen quality [108]. A more recent study However, additional studies are needed to determine
by Bujan et al. supports this conclusion; however, the if, at higher concentrations or longer duration of use,
authors did not find a correlation between duration of antiretrovirals themselves may lead to inhibited sperm
antiretroviral treatment and semen parameters [110]. function.
The era of highly active antiretroviral therapy
(HAART) has dramatically changed the lives of
patients with HIV, resulting in increased life expect- Leukocytospermia and male-factor
ancy and improved quality of life. With the advent of infertility
HIV sperm washing techniques, parenthood has been
achieved by male HIV patients using assisted repro-
Mediators of inflammation
ductive technology (ART). Sperm washing is based Immune cells present in human semen
on the principle that the HIV virus is found in seminal The testes are immunologically sequestered organs,
plasma and seminal leukocytes, but is not associated and as such represent an unusual deviation from the
with sperm [112,113]. Semen samples are centrifuged meticulous regulation imposed on the human body
multiple times using density gradient separation tech- by the immune system. This sequestration is achieved
niques, and this is followed by sperm swim-up proce- through Sertoli cells intimately linked by tight junc-
dures to isolate viable sperm [114]. The sperm isolate tions, forming the blood–testis barrier. Additionally,
is then tested for HIV by nucleic acid amplification the molecular milieu of the testicle may also result in
techniques, with Marina et al. noting HIV detection in immunological tolerance. Therefore, the presence of
6% of samples and, more recently, Persico et al. report- abnormal concentrations of leukocytes within the male
ing 0% detection rates [115,116]. In a comprehensive reproductive tract may represent a pathophysiological
review of the literature, Gilling-Smith et al. did not process contributing to male infertility (Table 17.6)
find any cases of seroconversion in female partners [4,118–133].
or children born in over 3000 cycles of sperm wash- Several groups have investigated WBC sub-
ing from HIV-infected males utilized in conjunction populations in the semen of fertile and infertile
with ART [117]. men (Table 17.7) [121,125,126,134]. El-Demiry and
With respect to ART, Nicopoullos et al. demon- colleagues used monoclonal antibodies (mAb) to
strated that the use of antiretrovirals and undetectable characterize the lymphocyte subpopulations along 307

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 Chapter 17: Genital tract infection and inflammation

Table 17.6. Prevalence of leukocytospermia among infertile men

Reference Detection Type of Threshold Prevalence n Sperm parameters
method WBC
Endtz, 1974 Peroxidase PMN 0.5×106/mL 24% 300 Not investigated
[118]
Comhaire, 1980 Peroxidase PMN 1×106/mL 13% 500 Not investigated
[4]
Jochum, 1986 PMN-elastase PMN 1000 ng/mL 19% 163 No effects on sperm count and
[119] morphology
Wolff, 1988 [120] PMN-elastase PMN 1000 ng/mL 15% 118 Not investigated
Wolff, 1988 [121] Immunohistology All 1×106/mL 24% 118 Not investigated
Wolff, 1990 [122] Immunohistology All 1×106/mL 23% 179 ↓Total sperm number, %
motility, sperm velocity, motility
index, total motile sperm
Gonzales, 1992 Giemsa/ All 1×106/mL 38% 280 ↓Sperm count,a % motile sperm,
[123] Papanicoulau % sperm vitality
Tomlinson, 1992 Immunohistology All 1×106/mL 5% 351 Not associated with reduced
[124] semen quality or conception
rates
Kung, 1993 [125] Immunohistology All 1×106/mL 2% 49 Inverse correlation to morph
and motility
Tomlinson, 1993 Immunohistology All 1×106/mL 3% 512 ↑ Ideal forms, ↓ head defects, ↓
[126] oligospermia
Wang, 1994 Immunohistology All 1×106/mL 8% 101 Not investigated
[127]
Yanushpolksy, Peroxidase PMN 0.5×106/mL 11% 1710 ↓ Conc
1995 [128] 6
1×10 /mL 7% –
6
2×10 /mL 4% ↓ Morph
Arata, 2000 [129] Peroxidase PMN 1×106/mL 32% 62 ↓ Conc, motility, HOS
Kaleli, 2000 [130] Peroxidase PMN 6
1×10 /mL 72% 219 ↑ Conc, HOS, acrosome reaction
Sharma, 2001 Peroxidase PMN 1×106/mL 9% 271 ↑ ROS
[131]
Saleh, 2002 [132] Peroxidase PMN 1×106/mL 33% 48 ↑ ROS, DNA damage,
↓ motility
Aziz, 2004 [133] Peroxidase PMN 1×106/mL 36% 56 ↓ Sperm structural integrity
Conc, concentration; DNA, deoxyribonucleic acid; HOS, hypo-osmotic swelling; Morph, morphology; PMN, polymorphonuclear
leukocyte; ROS, reactive oxygen species; WBC, white blood cell.
a
Only in the presence of hypofunctional seminal vesicles as assayed by fructose levels
(Adapted from Anderson DJ. The effect of genital tract infection and inflammation on male infertility. In: Lipshultz LI, Howards SS,
eds. Infertility in the Male, 3rd edn. St. Louis, MO: Mosby Year Book, 1997: 326–35.)

the genital tract of normal men and men with infer- Additionally, aggregates of leukocytes were seen in the
tility [135]. The authors demonstrated the presence of interstitium of the testis. This suggests that in normal
predominantly CD8+ suppressor/cytotoxic T cells in tissue, cellular mechanisms and an intact blood–testis
the epithelial lining of the prostate, seminal vesicle, barrier prevent immune responses to sperm antigens.
vas deferens, and epididymis. While no lymphocytes Wolff and Anderson characterized leukocyte subpopu-
were present in testis biopsies from normal men, all lations (granulocytes, monocytes/macrophages, CD4+
10 testicular biopsies from infertile men demonstrated T lymphocytes, CD8+ T lymphocytes, B lymphocytes)
T lymphocytes between the fibrous tunica propria and by mAb in the semen of 17 fertile and 51 infertile
308 the germinal epithelium of the seminiferous tubules. patients [121]. The study demonstrated that infertile

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Table 17.7. WBC subpopulations in semen of fertile and infertile men

Wolff, 1988 [121] Wolff, 1988 [121] Kung, 1993 [125] Kung, 1993 [125] Tomlinson, Aitken,
Mean(±SEM)/ Mean(±SEM)/ Mean(±SEM)/ Mean(±SEM)/ 1993 [126] 1994 [134]
ejaculatea n = 17 ejaculatea n = 51 ejaculatea n = 16 ejaculatea n = 51 Mean(±SEM)/mL Mean(±SEM)/
(fertile men) (infertile men) (fertile men) (infertile men) n = 229 (infertile mL n = 120b
men) (infertile men)
Total WBC 1 636 365 (4 966 260)c 7 199 090 (19 612 917) 385 708 (343 191)d 385 513 (997 398) 132 800 (26 900) 391 900 (90 500)
c
Granulocyte 1 348 160 (4 799 149) 5 407 075 (16 387 801) 261 562 (384 941) 343 159 (482 179) 67 000 (14 600) 396 900 (76 000)
Monocyte/macrophage 175 897 (288 476) 978 557 (1 788 724) 47 541 (146 258) 53 450 (137 152) 23 900 (3 200) 38 300 (20 100)
CD4+ T lymphocyte 9 205 (13 804) 155 505 (555 658) 52 066 (105 511) 52 020 (103 149) 940 (160) 60 600 (15 800) e
CD8+ T lymphocyte 6 250 (13 521)c 86 112 (211 047) 0 (0) 6 685 (26 637) 1170 (180)
B lymphocyte 2 737 (6 869) 88 634 (415 199) 28 427 (57 44) 13 838 (46 811) 1620 (460) 37 700 (9 800)
WBC, white blood cells.
a
WBC/ejaculate = ((WBC/field)/(sperm/field)) × (sperm/ejaculate).
b
Further differentiation of WBC subtypes was performed in 91 patients.
c
Wolff, 1988: significantly different between fertile and infertile groups by Kolmogorow–Smirnov test (P < 0.05).
d
Kung, 1993: No difference in any WBC appreciated between fertile and infertile groups.
e
Total T lymphocytes only, not CD4+ or CD8+ subpopulations.
(Adapted from Keck C et al. Seminal tract infections: impact on male fertility and treatment options. Hum Reprod Update. 1998; 4: 891–903.)

309
 Chapter 17: Genital tract infection and inflammation

Monocytes/
B-Lymphocytes
T-Lymphocytes

Granulocyte

B-cell/T-cell
Interaction
Cytokines

Plasma
Cell
ROS Elastase
OH H
2 O
2
O–2

Lipid
Peroxidation
DNA Anti-Sperm Abs
Damage

Spermatozoa Damage
and Dysfunction

Fig. 17.2. Paradigm for leukocyte subpopulation interaction in infertile men with leukocytospermia.

men had significantly more seminal leukocytes than velocity) [122]. Furthermore, Arata et al. found that
fertile men, with granulocytes representing the most in semen of infertile men, leukocyte concentration
prevalent subpopulation in both cases. Semen of infer- (peroxidase) was negatively correlated with sperm
tile men also demonstrated significantly more granu- morphology, motility, and hypo-osmotic swelling
locytes and CD8+ T lymphocytes. Finally, significantly [129]. Other investigators have noted adverse effects
more men in the infertile group than in the fertile group of leukocytospermia on semen parameters only in the
had leukocytospermia. In total, these studies suggest presence of seminal vesicle dysfunction (low fructose
that increased concentrations of leukocytes in the value corrected for sperm count) [123]. Aitken et al.
genital tract and semen are associated with infertil- noted that semen parameters were not negatively
ity. In Figure 17.2 we present a paradigm detailing the influenced by the presence of leukocytes; sperm func-
numerous mechanisms by which subpopulations of tion was only negatively impacted in washed sperm
leukocytes can disrupt normal spermatogenesis and preparations contaminated by leukocytes. The authors
sperm function. postulated that these effects may be attributable to the
Several authors have noted a negative correlation antioxidative properties of seminal plasma, miss-
between the presence of seminal leukocytes and other ing in the washed samples [134]. More recently, Aziz
semen analysis parameters. Wolff and colleagues et al. demonstrated that infertile men with leukocyto-
reported that infertile men with leukocytospermia spermia (peroxidase) had worse sperm motility and
(mAb) had significant reductions in total sperm morphology than both infertile men without leuko-
number and parameters of motility (percent sperm cytospermia and fertile controls [133]. The authors
310 motility, motility index, total motile sperm, and sperm also noted significant negative correlations between

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 Chapter 17: Genital tract infection and inflammation

leukocyte concentration, percentage sperm motility, morphology, motility, oocyte penetration, or acrosome
and various indices of morphology. reaction [134]. In two separate studies, el-Demiry and
Several authors have reported adverse effects of colleagues demonstrated that semen of fertile men
leukocytes on the sperm penetration assay (SPA). had higher concentrations of leukocytes and granu-
Berger et al. first reported that the concentrations of locytes than semen from sub/infertile men [138,139].
leukocytes, granulocytes, and lymphocytes all cor- The authors suggest that leukocytes in the semen may
related with abnormal SPA assay results (< 11% eggs represent both a nonspecific defense mechanism and a
penetrated) [136]. Maruyama et al. demonstrated that scavenging system for damaged, dead, and immature
in fertile donors the human spermatozoa penetration sperm. In a prospective study, Tomlinson et al. inves-
of zona-free hamster oocytes decreased in the presence tigated the effects of leukocytes on seminal param-
of the donors’ own plasma-purified leukocytes [137]. eters and pregnancy outcomes in 229 patients with
Interestingly, in infertile patients with more than 3 × 106 male-factor infertility [126]. The authors found that
leukocytes/mL, the penetration rates increased with although granulocyte and macrophage concentration
the removal of seminal leukocytes. In contrast to these had modest negative correlations with the percentage
studies, Aitken et al. found no correlation between the of spermatozoa head defects (r = –0.17, P < 0.001, and
concentration or total number of leukocytes and ham- r = –0.15, P < 0.01, respectively), these subpopulations
ster oocyte penetration in men attending an infertil- and total leukocytes were not associated with motility
ity clinic [134]. However, as briefly mentioned above, or sperm density. In fact, leukocytes were positively
leukocyte contamination of washed sperm prepara- correlated with percent “ideal forms” (morphology
tions significantly decreased penetration, suggesting a index). Furthermore, total granulocytes in semen
protective role of seminal plasma. were predictive of pregnancy (over a two-year period).
Granulocyte concentration in semen has been These results are corroborated by Kaleli et al., who
specifically investigated, since granulocytes are the demonstrated a positive association between leukocyte
predominant leukocyte subset in fertile and infertile concentration and sperm concentration and function
men with leukocytospermia [122,126,134,138,139]. [130]. In total, these findings suggest a potential pro-
Yanushpolsky and associates reported that in semen tective role of leukocytes, especially granulocytes, in
samples of 1710 male partners of infertile couples, sperm quality.
granulocyte concentrations (peroxidase) greater than
5 × 105 cells/mL and 2 × 106 cells/mL were associated Oxidative stress and reactive oxygen species
with significant differences in sperm concentration Oxidative stress is a fundamental pathophysiological
and morphology, respectively [140]. The authors also process mediated by oxygen and oxygen derivatives
found a robust correlation between the number of known as reactive oxygen species (ROS). Prominent
granulocytes and the percent of samples with less than examples of ROS include hydroxyl peroxide, super-
107 motile sperm per ejaculate (r = 0.94, P < 0.0001). oxide anion, hydroxyl radical, peroxyl radical, and the
These results are corroborated by Thomas et al., who hypochlorite radical. ROS are classified as free radical
demonstrated that in 79 male patients attending a fer- molecules because they harbor an unpaired electron.
tility clinic, seminal granulocyte concentrations (per- They are inherently unstable and highly reactive. ROS
oxidase) greater than 5 × 105 cells /mL and 1 × 106 cells/ serve normal physiological roles in cellular mes-
mL were both associated with a significantly lower saging systems. However, in pathological states, the
percentage normal sperm morphology than seminal inherent reactivity of ROS overwhelms endogenous
granulocyte concentrations below these values [141]. antioxidant systems and is the principal force medi-
The authors also noted a negative correlation between ating oxidative stress-induced cellular damage [142].
granulocyte concentration and normal morphology This process is well documented in a variety of disease
(r = –0.27, P < 0.01). states, including cardiovascular, autoimmune, and
The clinical relevance of leukocytospermia in infectious processes.
regard to male fertility is controversial, as other In the context of the testicular microenvironment,
reports argue against detrimental effects of leukocytes two cell populations are involved in the production of
on spermatozoa. In a study of 120 men attending an ROS: spermatozoa (especially damaged spermatozoa)
infertility clinic, Aitken et al. demonstrated that leuko- and seminal leukocytes – most notably granulocytes
cytospermia (WHO) had no influence on sperm [143–145]. Recently, immature spermatozoa within 311

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 Chapter 17: Genital tract infection and inflammation

the ejaculates of male subjects were also shown to pro- sperm dysfunction in washed spermatozoa prepara-
duce more ROS than mature spermatozoa. Some have tions, which are devoid of seminal plasma [134,143].
suggested that this finding may adversely impact male This observation is supported by studies of infertile
fertility potential as the immature sperm migrate from couples undergoing IVF in which leukocyte contami-
the seminiferous tubules to the epididymis [146,147]. nation of sperm preparations (i.e., lacking seminal
However, granulocytes are responsible for the major- plasma) resulted in elevated levels of ROS, decreased
ity of the ROS burden, contributing an estimated 103 sperm motility, and decreased fertilization capacity
greater magnitude than contributed by spermatozoa [148,159]. Several lines of investigation demonstrate
[148,149]. In regard to the pathogenesis of male-factor the protective role of seminal plasma against oxida-
infertility, oxidative stress results in lipid peroxidation tive stress-induced sperm dysfunction [153,155]. The
(LPO) of the sperm membrane with resultant mem- seminal plasma of infertile men also has lower levels
brane dysfunction and DNA damage. of total antioxidant capacity, lower levels of individual
Several studies have established that chemically antioxidants, and correspondingly higher levels of lipid
induced oxidative stress results in LPO of the sperm peroxidation than seminal plasma from fertile controls
membrane with abnormal changes in membrane com- [160–162].
position, significant impairment in sperm motility, As mentioned above, DNA damage results from
and disordered sperm–oocyte fusion [150–153]. ROS oxidative stress and, in turn, decreases the genetic via-
may decrease sperm motility through axonemal dam- bility of spermatozoa. Kodama et al. demonstrated that
age and depletion of ATP [154]. Furthermore, LPO spermatozoal DNA from infertile males demonstrated
appears to be significantly accelerated in damaged and greater oxidative damage than controls [163]. This is
defective spermatozoa [150,153]. Addition of seminal corroborated by basic science data from an in-vitro
plasma, endogenous protective enzymes (superoxide study of NADPH-induced generation of ROS demon-
dismutase, catalase), and antioxidants has been shown strating increased sperm DNA fragmentation [164].
to inhibit subsequent damage by LPO and, in certain Both studies also revealed beneficial effects from in-
cases, to reverse LPO-induced dysfunction in vitro vitro antioxidants on oxidative DNA damage, although
[150,153]. each study had varying results depending on the anti-
Similar results have been demonstrated using oxidant used and the source of the oxidative damage.
human spermatozoa incubated with ROS-producing As previously described, ROS production by immature
activated human granulocytes [148,149,155]. Studies spermatozoa may cause DNA damage in addition to
investigating semen samples from male patients increased LPO in mature spermatozoa during sperm
undergoing infertility evaluations demonstrated sig- migration [147,165]. Alvarez et al. examined male sub-
nificant associations between ROS production and jects with leukocytospermia and demonstrated a sig-
impaired sperm function [143–145,150]. A prospec- nificant increase in sperm DNA damage versus healthy
tive study by Aitken et al. revealed that ROS levels were donors and patients without leukocytospermia [165].
negatively correlated to both sperm–oocyte fusion Erenpreiss et al. also noted similar results, finding
(zona-free hamster oocyte penetration test) and in- that semen from infertile men with leukocytospermia
vivo fertility [156]. Zalata et al. demonstrated that in (peroxidase) and abnormal semen parameters was
semen of infertile men, leukocytospermia (peroxi- associated with increased DNA damage compared to
dase) was positively correlated to oxidative stress (r = semen from patients with leukocytospermia and nor-
0.79, P < 0.001) and negatively correlated to acrosin mal semen parameters [166]. This growing body of lit-
(enzyme involved in the acrosome reaction) activity erature suggests that leukocytospermia may increase
(r = –0.71, P < 0.001) [157]. Overall, an inverse cor- DNA damage in infertile men via ROS.
relation between oxidative stress and acrosin activity Several studies have directly demonstrated the det-
was also reported (r = –0.89, P < 0.001). Most recently, rimental effects of leukocyte production of ROS on
in a retrospective study of 132 patients, Agarwal et al. semen parameters and sperm function (Table 17.8)
demonstrated a significant association between ROS [131,132,134,143,165]. As mentioned previously,
levels (104 cpm/20 × 106 sperm) and male-factor infer- Aitken et al. demonstrated that leukocyte production
tility (OR = 4.25, P = 0.0034) [158]. of ROS inhibited oocyte fusion in sperm prepara-
Several investigators have reported that leuko- tions devoid of seminal plasma in 120 male patients
312 cyte-induced oxidative stress is only associated with attending an infertility clinic [134]. Furthermore, in a

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 Chapter 17: Genital tract infection and inflammation

Table 17.8. Association of leukocyte concentration with reactive oxygen species production in semen of infertile men
Reference n Leukocyte ROS assay Correlation Relevant findings
detection between
method leukocytes and
ROS
Aitken, 1994 120 Immuno- Chemiluminescence r = 0.78 (P < 0.001) In patients attending an infertility
[134] histochemistry in washeda sperm clinic, leukocyte contamination of
preparations washed sperm preparations resulted
contaminated by in decreased oocyte penetration
leukocytes
Aitken, 1995 115 Immunohisto- Chemiluminescence r = 0.729 (P < 0.001) In infertile subjects, leukocyte-
[143] chemistry in intact semen produced ROS were detected in
samples intact human semen samples, but
r = 0.840 (P < 0.001) no significant correlations were
in washed sperm noted between semen parameters
preparations (motility, morphology, sperm
concentration and total count)
or sperm function (penetration
assays) and either ROS or leukocyte
population. In infertile subjects,
leukocytes in washed sperm
preparations resulted in decreased
sperm motility, increased ROS, and
increased LPO versus controls
Sharma, 2001 271 Peroxidase test ROS–TACb r = –0.39 (P < 0.001) Oxidative stress increased with an
[131] increase in WBC count even when
WBC was much less than 1 × 106/
mL. Significant negative correlation
between oxidative stress and
sperm concentration, motility, and
morphology
Saleh, 2002 48 Peroxidase test ROS–TAC r = –0.56 (P < 0.001) Levels of spontaneous and induced
[132] ROS production in pure-sperm
suspensions (free of leukocytes)
from the infertile men with
leukocytospermia were higher
compared to infertile men without
leukocytospermia and to control
group
Alvarez, 2002 74 Peroxidase test Chemiluminescence r2 = 0.64 In patients attending an
[165] (P < 0.004)c infertility clinic, subjects with
leukocytospermia demonstrated
increased sperm DNA damage
versus subjects without
leukocytospermia or healthy donors
DNA, deoxyribonucleic acid; LPO, lipid peroxidation; ROS, reactive oxygen species; TAC, total antioxidant capacity; WBC, white blood
cells.
a
Free of seminal plasma.
b
ROS–TAC inversely correlated to oxidative stress, accounts for a negative correlation to leukocytes compared to
chemiluminescence.
c
Spermatozoa at a certain stage of maturation isolated by density gradient centrifugation.

subsequent study by Aitken et al. of 66 infertile males protects against leukocyte-produced ROS. It should be
and 49 healthy donors, the authors found that leukocyte noted that these studies employed chemiluminescence
production of ROS in unwashed semen samples was to assay ROS. More recently, Sharma et al. introduced
not correlated with seminal parameters or sperm func- the reactive oxygen species–total antioxidant capacity
tion [143]. These results suggest that seminal plasma (ROS–TAC) score, which measures sperm ROS and 313

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 Chapter 17: Genital tract infection and inflammation

seminal TAC chemiluminescence as well [167]. The may be present in the semen of infertile men with
composite ROS–TAC values are inversely correlated leukocytospermia.
with the level of oxidative stress. In a subsequent study Wolff et al. demonstrated that in 118 male patients
employing ROS–TAC, Sharma et al. demonstrated a with infertility, concentrations of granulocyte elastase
moderate correlation between the concentration of were positively correlated to white blood cell (WBC)
leukocytes (peroxidase) and ROS in 271 men attend- concentrations (measured by immunohistochemistry)
ing an infertility clinic [131]. A similar study examin- (r = 0.755) [120]. These results were corroborated by
ing infertile men with and without leukocytospermia studies finding elevated levels of granulocyte elastase
and healthy controls also noted a correlation between in infertile men with leukocytospermia [176,177].
leukocyte concentration (peroxidase) and oxidative Furthermore, Rajasekaran et al. found that granulo-
stress via ROS–TAC [132]. The authors noted that men cyte elastase was not significantly elevated in infertile
with leukocytospermia had decreased sperm motility, men without leukocytospermia compared to normal
presumably from LPO, and increased DNA damage – fertile controls, but was significantly elevated in infer-
both supporting the concept of cellular injury arising tile men with leukocytospermia compared to both of
from oxidative stress. these other two groups. These results were verified in
Leukocyte production of nitric oxide (NO) and a subsequent study by the authors, with an even more
other reactive nitrogen intermediates (RNI) consti- rigid definition of leukocytospermia (2 × 106 leuko-
tutes another chemical mediator system studied in cytes/mL) [178].
relation to sperm function. Spermatozoa are able to More recently, Zorn et al. found that granulocyte
produce NO for physiological functions [168,169], elastase in semen at a cutoff concentration of ≥ 290
but NO can also react with the superoxide anion form- ng/mL is an accurate predictor of leukocytospermia-
ing the highly reactive peroxynitrite (ONOO) anion, associated genital tract inflammation (sensitivity
resulting in cytotoxicity [170]. Toxicity from these 79.5%, specificity 74.4%) [179]. In findings similar
molecules is believed to arise from inhibition of cellu- to those of Wolff et al., the authors also noted a posi-
lar aerobic respiration, and has been demonstrated in tive but less robust correlation between granulocyte-
spermatozoa [171]. In-vitro studies revealed that incu- elastase concentration and leukocyte concentration
bating sperm with low concentrations (nM) of sodium in 312 infertile male patients (r = 0.330). Although
nitroprusside (NO donor) enhanced sperm motility Zorn et al. found no correlation between granulocyte
and viability and decreased LPO [172]. In contrast, elastase and ROS concentration, Henkel and Schill
high concentrations adversely affected sperm motil- did find a positive correlation between these two
ity [124,171,173]. Sperm incubations with other NO parameters in a study of 176 men attending an infer-
donors support the deleterious effects of NO [171,173]. tility clinic (r = 0.613, P < 0.0001) [180]. Zorn et al.
Nitrite is a stable end product of NO and has been used also demonstrated a negative correlation between
as an assay of NO production. Nobunaga et al. dem- granulocyte-elastase concentrations and sperm DNA
onstrated that semen from infertile men with leuko- damage (r = –0.194, P = 0.024) suggesting a possible
cytospermia (peroxidase) had significantly elevated unexpected protective role for granulocyte elastase
levels of nitrite compared to semen from infertile [179]. No association was found between semen
men without leukocytospermia, and semen from fer- parameters and granulocyte-elastase concentrations.
tile controls [174]. However, Revelli et al. reported Interestingly, the authors also noted a higher concen-
that semen samples from men seen at a fertility clinic tration of granulocyte elastase in men whose female
revealed no correlation between nitrite concentration partners had tubal damage than in men whose female
and leukocyte concentration (anti-CD45 antibody) or partners had no tubal damage.
sperm motility [175].
Cytokines
Elastase Cytokines are the principle molecular signals of
As previously discussed, leukocytes in semen from the immune system and are released by both lym-
infertile men may result in cellular spermatozoal dam- phocytes and monocytes. Certain cytokines activate
age through the release of ROS. Since granulocytes cellular proliferation and inflammatory mechanisms,
are the major constituent of seminal leukocytes, other ultimately resulting in oxidative stress, while oth-
314 mediators of granulocyte-induced cellular damage ers act in direct opposition to inflammation. If

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 Chapter 17: Genital tract infection and inflammation

leukocytospermia results in increased oxidative controls and infertile men without leukocytospermia
stress, then a preponderance of increased pro-inflam- [187,188]. Comhaire et al. also noted that IL-1β con-
matory and decreased anti-inflammatory cytokines centrations were elevated in men with leukocyt-
with resulting impairment of sperm function should ospermia when compared to those in controls [185].
be appreciated. In fact, Hill et al. demonstrated that Interestingly, TNF-α, IL-1α, and IL-1β were all shown
incubating spermatozoa with supernatant from to stimulate significant production of ROS by sperm
activated peripheral blood leukocytes significantly from fertile donors in an in-vitro setting [189].
decreased sperm motility [181]. Not all pro-inflammatory cytokines contribute to
Rajasekaran et al. noted that seminal plasma of fertility difficulties in males with leukocytospermia.
infertile men with leukocytospermia (Papanicolaou Naz et al. reported higher levels of IL-12 in fertile
stain and granulocyte-elastase assay) had significantly men than in infertile men with leukocytospermia
elevated levels of IL-2 and IL-8 compared to seminal [190] and infertile men without leukocytosper-
plasma of infertile men without leukocytospermia and mia [190,191]. IL-12, which is released by activated
of fertile controls. IL-2 activates and induces prolifera- macrophages, has no currently known role in leuko-
tion of T-helper cell lymphocytes; this suggests a pos- cytospermia-associated infertility. However, other
sible role for cell-mediated immunity. The authors also signaling molecules associated with monocyte activ-
noted that IL-8 but not IL-2 was significantly related ity, such as monocyte chemotactic and activating fac-
to ROS levels (chemiluminescence, r = 0.704). Since tor (MCAF) and tissue factor (TF), have been shown
IL-8 is a granulocyte chemoattractant, these results to be elevated in semen of infertile men with leuko-
strengthen the argument for leukocytospermia- cytospermia when compared with semen of infertile
induced ROS production. Other studies also demon- men without leukocytospermia and with that of fer-
strated increased levels of IL-8 in infertile men with tile controls [187,188].
leukocytospermia (mostly granulocytes) [182,183]. Lastly, certain cytokines antagonize the inflam-
Furthermore, Shimoya et al. demonstrated a significant matory process and serve to regulate the immune
correlation between and IL-8 and granulocyte-elastase response. Two such anti-inflammatory cytokines, IL-4
levels (r = 0.688, P < 0.001). and IL-10, were demonstrated to be significantly lower
Tumor necrosis factor α (TNF-α), IL-1, and IL-6 in infertile men with and without leukocytospermia
are all pro-inflammatory cytokines. TNF-α was found than in fertile controls [178,184].
to be elevated in infertile men with leukocytospermia The studies described suggest that pro-inflam-
compared to infertile men without leukocytospermia matory cytokines are increased in infertile men with
[182,184] and fertile controls [184]. It should be noted leukocytospermia and may impair sperm function,
that Maegawa et al. used granulocyte-elastase con- and they suggest an important role for both mono-
centrations greater than 1000 ng/mL to define leuko- cytes and lymphocytes in the pathophysiology of
cytospermia, whereas Omu et al. used Papanicolaou leukocytospermia. The release of cytokines from
staining and WHO criteria to measure and define leu- these cells activates the immune system, specifi-
kocytospermia. Hill et al. reported that spermatozoa cally triggering the proliferation and activation of
incubated with TNF-α and interferon γ demonstrated T lymphocytes, B lymphocytes, granulocytes, and
decreased motility [181]. Taken together, these stud- macrophages. Ultimately, a cascade of inflammatory
ies suggest that TNF-α is elevated in infertile men with changes leads to impaired sperm function.
leukocytospermia, and that this elevation leads to
impaired sperm function. Antisperm antibodies
Several other investigators also noted increased Antisperm antibodies (ASA) are present in 3–12%
concentrations of IL-6 in semen of men with leukocyto- of men seeking evaluation at infertility clinics. ASA
spermia (fertility status unknown) [185] and infertile interfere with fertilization via several mechanisms:
men [186] compared to those in fertile controls. Naz agglutination of sperm, prevention of cervical mucus
et al. showed that the elevation of IL-6 in infertile men penetration, and inhibition of sperm–oocyte interac-
was significantly correlated with detrimental effects on tion/fusion [192]. Production of ASA is postulated to
sperm number, motility, and penetration. Furthermore, result from three basic changes in the affected patient:
IL-6 concentrations were demonstrated to be greater namely, the breakdown of the blood–testis barrier,
in infertile men with leukocytospermia than in fertile inoculation of the host with sperm antigens, and failure 315

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 Chapter 17: Genital tract infection and inflammation

of immunosuppression. Infections of the genital tract, of semen samples of infertile patients [120,126]. In the
such as acute and chronic prostatitis, are well-known testicular microenvironment, the release of cytokines
risk factors for ASA generation [192]. However, the such as IL-1, IL-2, and IL-6 (discussed above), together
data regarding the risk associated with leukocytosper- with T-helper cells, may activate and differentiate these
mia are less well characterized. localized B cells into plasma cells and induce produc-
Wolff et al. noted no association between leuko- tion of ASA.
cytospermia (mAb) and ASA in 179 infertile patients
[122]. These results have been corroborated by sub- Detection of leukocytospermia
sequent studies [126,193,194]. Gonzalez et al. also Currently, the WHO defines leukocytospermia
demonstrated no overall association between leuko- as ≥ 1 × 106 WBCs/mL semen. This recommenda-
cytospermia and ASA [195]. However, the authors did tion is derived from Comhaire’s threshold of 1 × 106
note that in the setting of seminal vesicle dysfunction, peroxidase-positive leukocytes per mL semen for the
infertile patients with leukocytospermia had a sig- diagnosis of male adnexitis (male accessory gland
nificant increase in ASA compared with patients with inflammation) [4]. Several tests available to identify
leukocytospermia and normal seminal vesicle function and quantify seminal leukocytes are discussed below.
and patients without leukocytospermia. This suggests
a possible association between seminal vesicle func- Histology/morphology
tion and immune regulation. Round cell counts
Paschke and colleagues demonstrated in a cohort The concept of “round cells” is important when con-
of 1189 men evaluated for infertility that leukocyto- sidering leukocyte quantification. Round cells refer
spermia (peroxidase) was significantly correlated to to the similar morphology shared by both seminal
mixed agglutination reaction (MAR) IgG and IgA WBCs and immature germ cells when viewed under
results [196]. Other studies have reported a high phase microscopy. In a study of 108 males of infer-
prevalence of ASA in infertile men with leukocyt- tile couples, Eggert-Kruse et al. demonstrated that
ospermia [184,195]. Only Omu et al. distinguished a majority of round cells were immature germ cells,
between microbial and nonmicrobial (idiopathic) while less than 5% were WBCs (range 0–58%, median
causes of leukocytospermia, and the authors noted a 3%) [198]. Interestingly, Sigman and Lopes noted that
higher prevalence of ASA in men with microbial than in 627 men presenting for an infertility evaluation,
in those with non-microbial leukocytospermia. Both leukocytospermia was present in 35% of semen sam-
groups had a higher prevalence of ASA than fertile ples with excessive round cells (> 10/hpf or 1 × 106/
controls. mL) [23]. The authors concluded that the majority of
More recently, Eggert-Kruse et al. demonstrated patients with excessive round cells do not have leuko-
that IgA antibodies against human 60 kDa heat shock cytospermia. However, the two are not mutually exclu-
protein (HSP60) were significantly associated with sive. Quantification of round cells by phase microscopy
leukocytospermia (mAb) [197]. Heat shock proteins alone is not an accurate method for the determination
are chaperone proteins that respond to environmental of leukocytospermia.
stress and represent a cellular injury response mecha- Bryan–Leishman
nism. Antibodies against HSP may result in sperm that
The Bryan–Leishman stain was developed by Couture
are more susceptible to oxidative stress, thus potentially
et al. in 1976 [199]. Comparison of the Bryan–Leishman
contributing to infertility. Although Eggert-Kruse et al.
test with immunohistochemistry overestimates lym-
reported that the presence of anti-HSP60 antibodies
phocytes and underestimates granulocytes in semen
was not associated with abnormal semen parameters
smears [200].
or sperm function, HSPs may represent another target
of leukocytospermia pathology. HSP impairment may Peroxidase/Endtz
not be detected on routine semen analysis or sperm The peroxidase test, or Endtz test, was first described
function testing, but it may result in impaired intracel- by Endtz in 1974. This test stains for the peroxidase
lular responses to oxidative stress. enzyme present in granulocytes and thus measures
Humoral immunity mediated by T-helper and B only this subpopulation of the WBCs [118]. Because
lymphocytes (rather than granulocytes) drives the granulocytes are typically the most prevalent WBC
316 production of ASA, with B cells detected in 20–59% found in semen, the peroxidase test is clinically

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 Chapter 17: Genital tract infection and inflammation

useful and recommended by the WHO for granulocyte Products of leukocytes/inflammatory markers
detection [201]. Elastase
In a study of 87 men undergoing an infertility Granulocyte elastase is an enzyme specific to activated
evaluation and 25 controls, Potlich et al. demonstrated granulocytes, and it can be detected in fresh or frozen–
a robust correlation between peroxidase and immuno- thawed seminal plasma by immunoassay. Elastase
histology for WBC enumeration (r = 0.70, P < 0.0001) levels are generally categorized into three groups:
[202]. However, the authors noted that WBC concen- < 250 ng/mL = no inflammation; 250–1000 ng/mL =
trations as measured by immunohistology were sig- intermediate range; and > 1,000 ng/mL = genital tract
nificantly higher than those measured by peroxidase inflammation [119]. Wolff et al. found that elastase
testing. In fact, leukocytospermia was detected in 8.9% detection using ELISA correlated strongly with im-
of samples assayed by peroxidase, whereas this number munohistochemical means of granulocyte detection
rose to 15.2% of samples when immunohistology was [120]. Henkel et al. found a strong correlation between
employed. Overall, the peroxidase test had a sensitivity granulocyte elastase and ROS production (r = 0.613, P =
of 58.8% and a specificity of close to 100% when com- 0.0001) [180]. Granulocyte-elastase assessment results
pared to the “gold standard” of immunohistology. in an objective measure of activated seminal granulo-
Immunological techniques cytes, but the large cost associated with using ELISA
Immunohistology may deter its use. Furthermore, elastase indicates male
The “gold standard” for WBC detection and quantifica- adnexitis, and may therefore represent a better indica-
tion is immunohistology. Immunohistology employs tor of inflammation than seminal WBC counts.
monoclonal antibodies (mAb) targeted against WBC Chemiluminescence
surface markers [203]. For instance, antibodies directed Chemiluminescence (CL) is an indirect method for
against the common leukocyte antigen CD45 will leukocyte detection, as it quantifies leukocyte produc-
detect granulocytes, macrophages, and lymphocytes. tion of ROS. In its most basic form, CL involves a molec-
Additionally, mAb may be targeted against WBC sub- ular probe (luminol), which emits light in the presence
population-specific cell markers (e.g., CD15 detects of ROS. A luminometer measures and quantifies this
granulocytes). Visualization methods of mAb-stained signal, which is proportional to the amount of ROS.
specimens often employ direct color contrasts between Leino et al. demonstrated a high degree of correlation
positive and negative cells (e.g., positive-red vs. nega- (r = 0.932, P < 0.001) between CL and peroxidase meth-
tive-blue). Alternatively, immunofluorescence (mAb- ods for WBC detection [206]. Limitations of this tech-
stained cells emit a colored light) may be utilized [204]. nique include the fact that CL is based on the metabolic
Overall, immunohistology provides the most accurate products of viable cells only and the expense of a lumino-
means of leukocyte detection and quantification. meter, which may preclude widespread use of this assay.
Flow cytometry The above tests demonstrate the various methods of
Flow cytometry, when used in conjunction with mono- identifying leukocytospermia. The peroxidase test and
clonal antibodies, can provide rapid analysis of scant granulocyte-elastase test appear to be useful screening
WBC subpopulations without purification proce- methods for leukocytospermia detection. For more
dures. Leukocytes, monocytes, and granulocytes are careful evaluation of leukocytospermia, immunohis-
differentiated using the light-scatter properties of the tochemistry is a highly accurate, but more timely and
WBCs and the density of the leukocyte marker anti- costly, method of leukocyte detection.
body, CD45. CD45 has low affinity for granulocytes,
higher affinity for monocytes, and the highest affinity Treatment of leukocytospermia
for lymphocytes. Ricci et al. demonstrated a strong cor- Several treatment options exist to protect spermatozoa
relation between flow cytometry (using mAbs against from the deleterious effects of leukocytes and therefore
CD45 and CD53) and the peroxidase test (r = 0.619, P optimize semen quality. The four major pharmaco-
< 0.0001) and the elastase test (r = 0.542, P < 0.0001). logic approaches are antimicrobial therapy to treat
When compared with flow cytometry, the peroxidase clinical or subclinical infection, anti-inflammatory
test had 58.8% sensitivity and a 92.8% specificity in medications (such as COX-2 inhibitors), antioxidant
detecting leukocytospermia, while the elastase test had therapy to minimize associated oxidative stress, and
a 78.8% sensitivity and 75% specificity [205]. antihistamines to stabilize mast cells. 317

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 Chapter 17: Genital tract infection and inflammation

Antibiotics twice per day, plus instruction to ejaculate at least once

A great deal of evidence supports the use of antibiot- every three days for a month. The baseline frequency of
ics to reduce leukocytospermia. In addition to treat- ejaculation for all patients before initiation of the study
ing infection, the goal of therapy is to restore sperm was 5 ± 1.3 times (range 2–10) per month. The authors
quality via an increase in sperm velocity, sperm found that after one month of treatment, 56% of men
number, percent motility, and total motile sperm. in group 2 and 76% of men in group 3 achieved resolu-
However, the use of antibiotics is not without risk. tion of their leukocytospermia, compared to only 6.7%
Many antibiotics have been implicated in negatively in the group 1, the no-treatment group. Resolution was
affecting fertility status. Nitrofurans, macrolides, significantly higher for both treatment groups when
aminoglycosides, tetracyclines, and sulfa drugs have compared to group 1 by chi-square analysis (P < 0.05
all been linked to infertility. Schlegel et al. suggest that for group 2; P < 0.01 for group 3). While the authors
extrinsic factors, such as antibiotic use, may play a reported an increase in frequency of ejaculation from
role in the 10–15% of couples demonstrating primary 5 to 10 times per month for patients in group 3 during
infertility [207]. the treatment phase, they did not report any data on
Branigan et al. conducted a prospective cohort frequency of ejaculation for patients in either group 1
study of men experiencing unexplained infertility or group 2 during the treatment phase. Thus, frequency
of at least one year’s duration [208]. Using a Bryan– of ejaculation was not truly controlled for, which some-
Leishman stain, the authors identified those patients what limits our ability to draw conclusions regarding
with WHO-defined leukocytospermia. Subjects were this parameter.
then randomly assigned to either a nontreatment Omu et al. examined the effects of antibiotics on
group or a treatment group receiving 100 mg doxy- semen parameters, seminal total antioxidant activ-
cycline twice daily for seven days, followed by 100 mg ity, vitamins A and E, T-helper cytokines, and ASA in
once a day for 21 days. The female partner of each male infertile men with leukocytospermia (hematoxylin/
was treated simultaneously with 100 mg doxycycline eosin stain) and/or bacteriospermia [210]. Patients
twice daily for five days, followed by 100 mg once a day were identified as (1) leukocytospermic with seminal
for five more days. The males were advised to ejaculate bacteria, (2) leukocytospermic without any seminal
frequently (at least every third day). The authors found bacteria, and (3) fertile controls. Preceding treatment
a 53% pregnancy rate (over six months) in female part- with antibiotics, sperm concentrations and sperm
ners of leukocytospermic men who were responsive motility were significantly higher in fertile controls
to treatment. This pregnancy rate was significantly than in the two leukocytospermic groups. Also, total
higher than that of leukocytospermic men who did not antioxidant activity was highest in the control group
respond to treatment (6%), leukocytospermic men who (3.2 ± 1.4 mmol/L), lower in the purely leukocytosper-
did not receive treatment (6%), and even men without mic group (1.7 ± 1.3), and lowest in the bacteriosper-
leukocytospermia (12%). No statistically significant mic and leukocytospermic group (1.1 ± 0.3). Likewise,
differences in sperm concentration, sperm motility, or inflammatory T-helper cytokines (IL-8 and IL-2) were
percent normal sperm morphology were seen between significantly higher in men with bacteriospermia
the men who responded to treatment and the other and leukocytospermia than in controls, while anti-
groups, either before or after therapy. The authors con- inflammatory IL-4 was higher in controls. ASA were
cluded that leukocytospermia that responds to antibi- more prevalent in the group with bacteriospermia and
otic therapy is suggestive of a bacterial etiology [208], leukocytospermia than in the group with leukocyto-
and this subgroup of leukocytospermic patients enjoys spermia only. Patients in group 1 received antibiotic
markedly improved pregnancy rates with antibiotic therapy, and following treatment the authors found
treatment. significant improvement in sperm concentration and
Yamamoto et al. examined 263 males attending motility. Antibiotic therapy also improved total anti-
an infertility clinic. Of these patients, 48 co-expressed oxidant activity (P < 0.02) and reduced IL-2 (P < 0.03)
both > 10 WBCs/hpf on EPS and > 1 × 106 WBCs/mL in and IL-8 (P < 0.05) levels, but increased IL-4 (P < 0.01).
semen [209]. These men then underwent randomiza- No differences in ASA levels were found. The authors
tion into either (1) no treatment, (2) trimethoprim 80 concluded that antibiotic therapy benefits affected
mg, sulfamethoxazole 400 mg orally twice per day, or (3) patients by reducing pro-inflammatory cytokines and
318
trimethoprim 80 mg, sulfamethoxazole 400 mg orally oxidative stress.

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 Chapter 17: Genital tract infection and inflammation

Though these trials do show beneficial effects of morphology. This suggests that while antibiotics may
antibiotics on the resolution of leukocytospermia, help improve seminal WBC concentrations, the treat-
improvement in semen parameters, and increased ments do not lead to improved semen parameters in
pregnancy rates, some important points should be asymptomatic leukocytospermic males.
noted. The trials used basic histology, not peroxidase The studies by Yanushpolsky et al. and Erel et al.
or mAb staining methods, to identify leukocytes. Also, both employed peroxidase staining of leukocytes
the etiology of leukocytospermia is of importance (i.e., and were structured as randomized controlled trials.
infection, abacterial, autoimmunity). Branigan et al. Overall, these two studies demonstrate better method-
did not distinguish between men with or without bac- ology than the studies advocating the use of antibiot-
teriospermia, while Omu et al. treated only those with ics. Furthermore, the choice of antibiotics is another
leukocytospermia and bacteriospermia and not those important issue that must be considered when review-
with leukocytospermia alone. ing these studies. Most of the above trials employed a
Yanushpolsky et al. conducted a prospective ran- predetermined, empiric treatment regimen, whereas
domized controlled study of leukocytospermic males Omu et al. used culture sensitivities to guide therapy.
and their partners who together had experienced unex- In summary, the efficacy of antibiotic treatment for
plained infertility and no evidence of genital infec- isolated leukocytospermia remains in question.
tion [128]. Identification of leukocytospermia was
conducted in accordance with WHO standards using COX-2 inhibitors
peroxidase testing. Subjects were randomly assigned Recently, Lackner et al. conducted a prospective non-
to groups in which: (1) patients and their partners randomized study examining the use of cyclooxygen-
received 100 mg oral dose of doxycycline twice per day ase-2 (COX-2) inhibitors in 12 patients with abacterial
for 14 days, (2) patients and their partners received tri- leukocytospermia [212]. COX-2 inhibitors are anti-
methoprim 160 mg/sulfamethoxazole 800 mg orally inflammatory medications that inhibit prostaglandin
twice per 14 days, or (3) patients received no therapy. production. The patients were treated with valdecoxib
The authors demonstrated no statistically significant 20 mg/day for two weeks. Following treatment, the
differences in the WBC count between the two groups authors found a reduction in leukocyte concentration
treated with antibiotics. Furthermore, Yanushpolsky from 5.5 × 106 WBCs/mL (interquartile range 3.3–6.8)
et al. found that semen parameters did not statistically to 1.0 × 106 WBCs/mL (interquartile range 0.3–2.0) (P
improve after antibiotic treatment; this led the authors < 0.001). Furthermore, sperm count was significantly
to conclude that antibiotic treatment does not provide increased, though motility and morphology did not
benefit to asymptomatic leukocytospermic males. improve. Though the study was small, these results
Erel et al. conducted a randomized controlled suggest a beneficial role for COX-2 inhibitors in
trial of 70 male partners of infertile couples [211]. patients with abacterial leukocytospermia.
After detection of leukocytospermia (> 1 × 106 WBCs/
mL) by peroxidase staining, subjects were randomly Antioxidants
assigned to one of three groups: (1) a placebo group, Vicari et al. conducted a prospective study of men
(2) a group that received 100 mg of doxycycline twice with chronic, abacterial prostatovesiculoepididymitis
a day for 10 days, and (3) a group that received 100 mg (PVE) [213]. Concurrently, subjects were also experi-
of doxycycline twice a day for 10 days plus two injec- encing infertility of a median duration of seven years,
tions of 0.5 g ceftriaxone intramuscularly. The authors as well as persistent leukocytospermia according to
found no statistically significant differences between WHO standards. Subjects were randomly assigned
the three groups for age, duration of infertility, sperm to one of four groups: (1) carnitine therapy for four
concentration, sperm motility, or sperm morphology months, (2) nonsteroidal anti-inflammatory drugs
at baseline. Following treatment, groups 2 and 3 had (NSAIDs) for four months, (3) NSAIDS for two
statistically significant reduction of WBC counts com- months followed by carnitine therapy for two months,
pared to baseline. However, there was no statistical and (4) NSAIDs simultaneously with carnitines for
difference in the resolution of leukocytospermia cases four months. Following treatment, the authors dem-
between treatment groups and the control group over onstrated improved sperm viability (%) and reduced
a four-week period. Furthermore, antibiotic treatment seminal leukocyte concentration in all groups com-
did not improve sperm concentration, motility, or pared with pretreatment values. Interestingly, group 3 319

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 Chapter 17: Genital tract infection and inflammation

not only had the highest percentage of viable sperm but dysfunction, abnormal semen composition, and leu-
was also the only treatment group to demonstrate sig- kocytospermia [218].
nificantly increased forward motility (%). These effects Aird et al. compared the semen of nine men with
observed in group 3 were complemented by the highest chronic SCI (> 1 year) obtained via electroejacula-
reduction of ROS production. Concomitant use of car- tion (both antegrade and retrograde) to semen from
nitines alongside NSAIDs in group 4 was less effective healthy donors [219]. Using monoclonal antibodies
at reducing ROS. Lastly, and perhaps most importantly, against leukocytes, the authors concluded that the
group 3 had the highest pregnancy rate (P < 0.01). increased percentage of leukocytes in semen of males
While the literature regarding carnitine use in male with SCI was the result of concomitant UTIs, espe-
infertility has been disappointing overall, this study cially in retrograde samples. Furthermore, impair-
suggests the use of carnitine following pretreatment ment of motility was not correlated with the number
with NSAIDs helps to optimize semen parameters, of leukocytes. Finally, granulocytes and macrophages
possibly by decreasing ROS. Additional studies need were the predominant leukocyte subtypes, and the
to be undertaken to corroborate the above findings. proportion of leukocytes in the semen of males with
SCI was not related to level, extent, or duration of SCI.
Antihistamines It should be appreciated that quality of retrograde
Recently, Olivia and Multigner conducted an open, semen samples is considered inferior to that of ante-
noncontrolled study of 55 men identified as having grade samples [220,221]. In a similar study, Trabulsi
leukocytospermia (> 1 × 106 WBCs/mL) by Giemsa et al. compared antegrade semen samples (electro-
staining [214]. After a 12-week course of ketotifen, ejaculation) of 17 males with SCI free of UTIs to age-
these men were found to have reduced seminal WBC matched controls [222]. The authors reported that the
counts. Ketotifen is an antihistamine-like drug with semen of men with SCI had significantly elevated total
mast cell stabilizing effects that inhibits WBC degranu- WBC-to-sperm ratios, granulocyte-to-sperm ratios,
lation and the release of inflammatory mediators. After and macrophage-to-sperm ratios compared to semen
four weeks of ketotifen at 1 mg twice a day, the authors of the control population. While this study demon-
reported a reduction of WBCs from 5 × 106 WBCs/mL strated increased leukocytes in the semen of male SCI
(interquartile range 2–6) to 0 WBCs/mL (P < 0.0001). patients in the absence of UTI, the effects of increased
Following therapy, sperm motility and sperm mor- leukocytes on semen parameters and sperm function
phology significantly improved from pretreatment were not assessed. Basu et al. also examined antegrade
values. Olivia and Multigner did not find any differ- semen samples from 12 male patients with SCI free of
ences between seminal volume, sperm concentration, UTIs compared with samples from eight healthy age-
and sperm count from ketotifen treatment after the matched controls [223]. Unlike investigators in the
12-week therapy. previous studies, the authors used penile vibratory
stimulation, not electroejaculation, for semen collec-
Spinal cord injury and leukocytospermia tion in men with SCI. Electroejaculation has been asso-
Every year there are approximately 10 000 new cases of ciated with decreased semen quality [217,220]. Flow
spinal cord injury (SCI) in the United States; the major- cytometry was employed to detect seminal leukocytes.
ity of victims are males in their reproductive years The authors found similar sperm concentrations in
[215]. SCI results in impaired sexual function and fer- both groups, but impaired sperm motility and viabil-
tility due to erectile dysfunction, ejaculatory dysfunc- ity in the semen of men with SCI when compared with
tion, and poor semen quality. Regardless of the level that of controls. Men with SCI also had significantly
and extent of SCI, semen analyses of male patients increased leukocytes, which were predominantly acti-
with SCI generally demonstrate normal sperm count vated T-helper lymphocytes. Mature granulocytes
with decreased motility and viability [216,217]. were also significantly elevated in the semen of men
Though considerable debate exists as to the etiology with SCI.
of sperm impairment, the observed adverse changes As mentioned previously in this chapter, leukocyte-
are likely multifactorial and secondary to stasis of induced damage of sperm may be mediated by ROS.
semen/sperm, scrotal hyperthermia, UTIs, ejacula- Interestingly, de Lamirande et al. demonstrated that
tory reflux of urine, SCI-related testicular atrophy, men with SCI have higher levels of seminal ROS than
320 SCI-related hypothalamic–pituitary–gonadal axis infertile men without SCI and healthy controls [154].

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 Chapter 17: Genital tract infection and inflammation

Furthermore, there was an inverse correlation between However, patients with CBP and NBP had significantly
percentage motility and ROS in men with SCI. These increased leukocytes (peroxidase) [229] (P < 0.001).
data are corroborated by Padron et al., who demon- Since the introduction of the NIH classification,
strated that men with SCI had decreased sperm motil- several authors have revisited this topic. Much of this
ity, impaired sperm morphology, increased seminal research has focused on type III prostatitis or CP/CPPS.
leukocytes (peroxidase test), and elevated ROS (chemi- Pasqualotto et al. demonstrated that men with type III
luminescence) when compared with healthy controls prostatitis had elevated seminal oxidative stress (ROS–
[224]. The levels of seminal ROS in men with SCI were TAC) versus controls [230]. Furthermore, sperm motility
also negatively correlated with sperm motility (r = was impaired only in type III patients with leukocyt-
–0.46 to –0.49, P = 0.02). Together, these studies sug- ospermia (peroxidase). Menkveld et al. demonstrated
gest an important role for ROS in leukocyte-mediated no difference in sperm concentration, motility, viability,
sperm damage in men with SCI. and WHO morphology criteria between patients with
Collectively, the studies suggest that abnormal type III prostatitis and controls [231]. However, further
concentrations of leukocytes are a common finding morphological investigation demonstrated that men
in the semen of men with SCI. While the etiology of with type IIIA had significantly elevated, elongated sper-
leukocytospermia is unclear in these men, the elevated matozoal forms compared to those of controls. Taken
concentration of WBCs may have a detrimental effect together, elevated oxidative stress secondary to prostatic
on sperm motility and viability through the produc- inflammation may be one mechanism for prostatitis-
tion of ROS. Recently, Randall et al. reported that the associated infertility. This is especially relevant for type
prostates of men with both SCI and leukocytospermia IIIA prostatitis. However, Ludwig et al. found no asso-
do not harbor increased inflammatory changes, sug- ciation between leukocytospermia (peroxidase) and
gesting that the prostate is not the source of seminal impaired semen parameters in men with type III prosta-
leukocytes [225]. Indeed, other genital tract changes titis [176]. A more recent study by Motrich et al. suggests
related to SCI may be driving the leukocyte response. that an autoimmune response to prostatic antigens may
result in infertility in men with type III prostatitis [232].
Further research is needed to delineate the relationship
Prostatitis, male infertility, and between CP/CPPS and male infertility.
leukocytospermia
The effect of prostatitis on male fertility is controversial. Conclusion
Much of this confusion is due to different classification In this chapter, we set out to provide a state-of-the-art
systems and methods of diagnosis employed in various overview of the impact of inflammation and infection
studies. Prior to the NIH classification system, several on male reproductive function. The complex relation-
studies demonstrated decreased semen parameters ship between infection, inflammation, and male fer-
in men with CP/CPPS. Christiansen et al. reported tility has been incompletely elucidated to date. The
decreased sperm concentration, motility, morphology, literature clearly demonstrates that many organisms are
and viability, and increased incidence of leukocyto- pathogenic to male reproduction, sometimes rendering
spermia, in men with chronic abacterial prostatove- men severely and permanently debilitated in terms of
siculitis versus controls (P < 0.001) [226]. However, fertility potential. Numerous studies have also shown
leukocytospermia was not predictive of abnormal that monocyte and T-lymphocyte release of cytokines
semen quality. These data are corroborated by Lieb et stimulates B-cell production of ASA and granulocyte
al., who also noted decreased sperm motility and mor- production of ROS and elastase, with detrimental
phology, and increased WBC concentration (Giemsa effects on semen quality and sperm function. Patients
stain), in patients with CP/CPPS (NBP and Pd) ver- with infection and inflammation of the genital tract are
sus controls (P < 0.05) [227]. In comparison to Leib, quite heterogeneous in terms of underlying etiologies
Krieger et al. examined differences in semen param- and responses to therapy. While many excellent studies
eters between patients with NBP and Pd. The authors describing treatment outcomes with various therapies
only noted decreased sperm motility in patients with have been reported in this chapter, additional prospec-
NBP versus those with Pd [228]. Conversely, Weidner tive, randomized clinical trials with clearly defined
et al. did not report a difference in semen param- patient populations are needed to help clarify which
321
eters among patients with CBP, NBP, Pd, or controls. treatments are truly beneficial and cost-effective.

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 Chapter 17: Genital tract infection and inflammation

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