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- Original Article -
Age-related changes in seminal polymorphonuclear elastase
in men with asymptomatic inflammation of the genital tract
Ralf Henkel1, Gesa Maab2, Andreas Jung2, Gerhard Haidl3, Wolf-Bernhard
Schill2, Hans-Christian Schuppe2
1Department of Medical Biosciences, University of the Western Cape, Bellville 7535, South Africa
2Centre for Dermatology and Andrology, Justus Liebig University, 35385 Giessen, Germany
3Department of Dermatology, Rheinische Friedrich-Wilhelms University, 53105 Bonn, Germany
Abstract
Aim: To investigate age-related inflammatory events in the male genital tract.
Methods: In a total of 4 265 randomly collected patients attending the andrological outpatient clinic of the Center for Dermatology and Andrology, University
of Giessen, Germany, ejaculate volume, pH-value, sperm concentration, total and progressive sperm motility,
concentration of polymorphonuclear (PMN) elastase, number of peroxidase-positive cells and fructose were measured
and correlated with patient's age. Results:
While ejaculate volume, motility and fructose all correlated negatively
with age, sperm concentration, PMN elastase and the pH-value showed a positive correlation. The prevalence of male
genital tract inflammation (as defined by PMN elastase > 250 ng/mL) and its severity increased significantly. PMN
elastase did not correlate with sperm motility. Fructose as a marker of seminal vesicle function showed a significant
negative relationship with the PMN elastase levels, the number of peroxidase-positive cells and sperm motility.
Conclusion: The significant increases of PMN-elastase levels as marker of male genital tract inflammation in older
men appear to be indicative of age-related changes in local immunoregulatory mechanisms. Because there is no
association of PMN elastase with sperm motility, a direct inhibitory effect of this enzyme can be
excluded. (Asian J Androl 2007 May; 9: 299_304)
Keywords: aging men; male genital tract inflammation; polymorphonuclear elastase; leukocytes; infertility; human semen
Correspondence to: Prof. Ralf Henkel, Department of Medical Biosciences, Private Bag X17, Bellville 7535, South Africa.
Tel: +27-21-959-2332 Fax: +27-21-959-3125
E-mail: rhenkel@uwc.ac.za
Received 2007-01-08 Accepted 2007-02-02
DOI: 10.1111/j.1745-7262.2007.00275.x
1 Introduction
Male genital tract inflammation is considered a major contributing factor to infertility. The majority of patients,
however, do not notice any clinical symptoms. Therefore, an accurate andrological examination according to World
Health Organization (WHO) guidelines is mandatory [1]. To prove genital tract inflammation and respective semen
patterns, several diagnostic methods have been described, including the determination of peroxidase-positive cells in
the ejaculate [2] or seminal polymorphonuclear (PMN) elastase [3]. However, the absence of leukocytes in semen
does not exclude the possibility of infections or inflammatory reactions of the male genital tract. Thus, the criterion
"leukocytospermia" as defined by WHO (more than
106 leukocytes/mL ejaculate) is not accurate and is still a matter of
ongoing debate [4, 5]. Even low numbers of macrophages in semen may indicate inflammatory disorders of the
epididymis and significantly impaired sperm functions and thus fertility [6]. In order to improve semen analysis in this
respect, the immunologic measurement of PMN elastase
was introduced [3] and has been proven a reliable marker
of silent male genital tract inflammation [7, 8].
Aging in men does not only decrease serum testosterone levels [9], but may also affect fertility [10, 11].
Moreover, the human immune system undergoes age-related changes that have been associated with increased
morbidity and mortality as a result of infections,
malignancies, or autoimmune diseases [12]. Notably,
immunosenescence is a complex process involving
multiple reorganizational and developmentally regulated
changes, rather than a simple unidirectional decline in all
functions [13]. One of the basic mechanisms proposed
to explain these aging processes is the free radical theory
[14]. It appears that almost every component of the
immune system undergoes age-dependent changes resulting in an imbalance between oxidative stress and
antioxidative defense, which will consequently lead to an
increased production of reactive oxygen species (ROS)
by phagocytes [12].
Because there are initial reports indicating increased
inflammatory events in the human genital tract in aged
men [7, 15] and spermatozoa are particularly susceptible
to oxidative stress caused by leukocytes, it was the aim
of this study to get further insight into the possible
mechanism of decreasing reproductive functions in older men.
2 Material and methods
In this retrospective study, a total of 4 265
consecutive patients attending the andrological outpatient clinic for
fertility problems between January 1998 and January 2000
(17_66 years old) were analyzed for the following parameters: age
(n = 3 916), volume of the ejaculate (n
= 4 121), pH-value (n = 4 113), sperm concentration
(n = 3 916), total motility (n = 4 265), progressive motility
(n = 4 265), concentration of the polymorphonuclear
granulocyte elastase (PMN elastase) (n = 2 115), number of
peroxidase-positive cells (n = 1 507) and the
concentration of fructose (n = 1 488) as marker of seminal vesicle
function. A complete set of data of all parameters was
available for 1 942 patients, which includes up to five
consecutive measurements per patient.
Semen analysis including the above-mentioned parameters was performed according to WHO guidelines
[2]. PMN elastase was determined in cell-free seminal
plasma as described by Jochum et al. [3], using an
enzyme-linked immuno-absorbent assay provided by DPC
Biermann (Bad Nauheim, Germany). Patients whose
ejaculates showed PMN elastase concentrations >
250 ng/mL were regarded as having a genital tract inflammation and
those showing PMN elastase levels >1 000 ng/mL as
having a severe inflammation [3].
All statistical calculations (mean, median and Spearman's Rank correlation) were performed after
testing for normal distribution of the data by means of the
Kolmogorov-Smirnov test. In addition, the data set was
categorized in three subgroups according to age (subgroup 1,
¡Ü 30 years; subgroup 2, 31_45 years; subgroup 3, > 45 years), medians and the differences
between the subgroups were calculated by means of the
Mann-Whitney U-test and H-test according to
Kruskal-Wallis. All calculations were carried out with MedCalc
(Version 9.2, MedCalc Software, Mariakerke, Belgium).
P < 0.05 was considered as significantly different.
3 Results
The summary statistics of all analyzed parameters
are compiled in Table 1. The high variation of values,
reflected by the high standard deviation (SD), is obvious
as it is expected for biological parameters. In Table 2,
the age of the patients is correlated with the results of
basic semen analysis and biochemical parameters.
Significant decreases of the values for the ejaculate volume,
motility, and for the concentration of fructose in elderly
men was revealed and the decline confirmed by means of
the H-test according to Kruskal-Wallis (P < 0.004) (Table
2). The decreases per year ranged between _0.70% for
total motility and _0.84% for the ejaculate volume.
However, significantly increasing values in older men were
found for sperm concentration, PMN elastase and the
pH-value, while the number of peroxidase-positive cells
remained unchanged (Table 2). The general prevalence
of male genital tract inflammation as determined by means
of PMN elastase (> 250 ng/mL) and the peroxidase stain
of leukocytes (> 106/mL) was 30.1% and
36.7%, respectively.
When focusing on male genital tract inflammation,
not only a significantly higher prevalence was obvious in
men older than 30 years, but also the seminal
concentration of PMN elastase increased (Table 3). The
concentration of PMN elastase between subgroups 1, 2 and 3,
respectively, differed significantly (Figure 1). However,
although PMN elastase and the number of
peroxidase-positive cells correlated significantly in an overall
analysis (n = 884; r = 0.649; P
< 0.0001), age-related changes in the number of peroxidase-positive cells could not be
observed (Table 2). In addition, significant negative
correlations were found for the parameters of inflammation
with the ejaculate volume (Table 4). The pH-value, which
is also indicative of male genital tract inflammation,
correlated positively with PMN elastase (n = 2 111;
r = 0.168; P < 0.0001) and the number of peroxidase-positive cells
(n = 1 507; r = 0.060; P = 0.0200), although the latter
association was markedly weaker. Both, PMN elastase
and the number of peroxidase-positive cells showed no
relationship with total motility (n = 1 773;
r = 0.029; P = 0.2191 and n = 1 380;
r = 0.002; P = 0.9442) and progressive motility
(n = 1 767; r = 0.004; P = 0.8590 and
n = 1 383; r = _0.002; P = 0.9462) respectively. This
result for the correlation between PMN elastase and
motility was not only obvious for all measurements
performed but also for all single patients with repeated
measurements (n = 264) (data not shown).
The functional marker of the seminal vesicles, fructose, showed a significant negative relationship with
PMN elastase (n = 1 291; r = _0.055;
P = 0.0495) and the number of peroxidase-positive cells
(n = 619; r = _0.211; P < 0.0001), but a weak positive correlation with the pH
(n = 1 487; r = 0.070; P = 0.0072) and the ejaculate
volume (n = 1 487; r = 0.222; P
< 0.0001). Its correlation with total (n
= 1 213; r = _0.061; P = 0.0336) and
progressive motility (n = 1 216; r = _0.062;
P = 0.0301) was negative.
4 Discussion
The age-dependent decrease of male reproductive
functions accompanied by a significant decrease of the
ejaculate volumes is a well-known phenomenon that is
caused by the significant decline of the concentration of
free testosterone and has been repeatedly described [9,
10]. With regard to genital tract infection or
inflammation among aging males, however, there are only two
short reports describing an age-dependent increase of
male genital tract inflammation as determined by seminal
PMN elastase [7] or WHO criteria of "male accessory
gland infection" including positive aerobic or anaerobic
bacteriological culture [15] in relatively small populations
of 312 and 388 patients, respectively.
Infection and inflammation of the male reproductive
tract are accepted as important etiological factors of male
infertility [1]. It should be noted, however, that a
correct diagnosis is hampered by imprecise definitions and
the asymptomatic course of these disorders in the
majority of patients. Thus, the reported prevalence of
genital tract inflammation varies considerably and is, of
course, dependent on the method and the cut-off values
used [5]. However, immunologic measurement of PMN
elastase has been proven a reliable marker of silent male
genital tract inflammation [3, 7, 8].
In this report, a large population study of 1 942
patients in which PMN elastase was analyzed and could be
correlated to other parameters, we can clearly confirm
the age-dependent increase of the prevalence of male
genital tract inflammation reflected by seminal PMN
elastase concentrations. The overall frequency of male
genital tract inflammation as detected by PMN elastase
levels higher than 250 ng/mL [3] and leukocytospermia
(>1 million leukocytes/mL ejaculate; [2]) was 30.1% and
34.6%, respectively. While leukocytospermia was more
frequent than that in previously published reports, the
prevalence of elevated PMN elastase concentrations in
semen corresponded with recent reports [2, 3, 7, 8]. In
addition to the increasing incidence of the inflammation,
we found an increased severity of male genital tract
infections in older men.
Despite a strong positive correlation of the PMN
elastase with the number of peroxidase-positive cells in
the ejaculate, the latter did not show any relationship to
age. Thus, the increase of PMN elastase in older men
might be explained by the observed age-related decrease
of the ejaculate volume, which has also been reported
previously [10]. Considering that the seminal vesicle
secretions contribute most to the ejaculate volume, its
decline can be due to an age- and therefore
testosterone-dependent seminal vesicle insufficiency [16] and would
be consistent with the negative relationship of fructose
as marker of seminal vesicle function with age.
The increased levels of PMN elastase in elderly men
can also indicate inflammation of the male accessory
glands [17], thus leading to reduced ejaculate volumes.
This hypothesis would be in agreement with our
observation that seminal volume is negatively correlated with
all parameters of inflammation investigated. Consistently,
fructose showed an inverse correlation with PMN elastase
and the number of peroxidase-positive cells, suggesting
an adverse effect of inflammation on the seminal vesicle.
These results also confirm data reported by Wolff
et al. [18].
However, the age-related increase of the seminal
concentration of PMN elastase might reflect changes in
local immune mechanisms as part of the general immunosenescence. Dysfunction of the aging immune
system comprises adaptive immune responses and innate defence mechanisms, and is thought to contribute
to an increased incidence of infections and chronic
inflammatory disease [7, 12, 15]. At the cellular level,
lymphocytes, phagocytes, and other immune cells undergo age-related alterations of their functions. For
example, the production of pro-inflammatory cytokines
is significantly increased [19]. Thus, it would be
plausible that the prevalence of male genital tract infections
and/or inflammation increases with aging.
Notably, immune cells including macrophages, mast
cells, and lymphocytes and their products are not only
encountered in the testis but also regular components of
the epididymis and excurrent ductal system [20].
Age-related changes in leukocyte activity might contribute to
high levels of the PMN elastase in seminal plasma. In
addition, inflammatory deterioration of accessory gland
function with decreased secretions could result in elevated
levels of the enzyme in the first instance. In addition, the
aforementioned age-related change in leukocyte activity
might contribute to the high levels of enzyme in seminal
plasma.
The deleterious influence of male genital tract
infections/inflammation and thus leukocytes on the excurrent
ductal system [21] and sperm functions like motility,
acrosomal function or sperm DNA fragmentation [22,
23] must not be underestimated. However, a direct
influence of this enzyme on sperm function seems rather
unlikely as PMN elastase appeared to have no influence
on sperm motility. This observation is consistent with
data reported by Maegawa et al. [24] who suggested
that a sufficient amount of the secretory leukocytes
protease inhibitor in seminal plasma prevents spermatozoa
from being attacked by elastase. Therefore, other
leukocyte-derived inflammatory factors like ROS or
pro-inflammatory cytokines [25] should also be considered
with regard to their impact on organ and sperm functions.
In contrast, Kopa et al. [8] reported a significant
negative correlation of seminal PMN elastase concentrations
and sperm motility.
In fact, the negative influence of peroxidase-positive
cells on progressive motility and sperm DNA
fragmentation seems to be mediated by ROS as reported
previously [5]. Thus, the age-related decrease in sperm
motility is not necessarily associated with the increased
seminal concentration of PMN elastase, but rather linked to a
dysfunctional epididymis in older men. The latter causes
a disturbed removal of the element zinc from the sperm
flagella during epididymal maturation, eventually leading
to poor motility [11]. Alternatively, an increased
imbalance between oxidative stress on the one hand, and a
lack of antioxidant capacity on the other hand, could cause
or at least contribute to the documented declines in sperm
function.
In conclusion, we found a significant increase of
PMN elastase levels in older men while the number of
peroxidase-positive cells in the ejaculate did not change,
which is most probably related to immunosenescence.
Because there is no association of PMN elastase with
sperm motility, a direct effect of this enzyme can be
excluded. Consequently, the age-dependent decrease of
sperm motility is most probably associated with altered
epididymal zinc elimination from the spermatozoa as
proposed in an earlier study [11], or ROS might play a role
in the pathological mechanism. This is the subject of
further investigation.
Acknowledgment
The authors appreciate the excellent linguistic review
by Ms S. Henkel and Ms B. Admin.
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