ORIGINAL ARTICLES: ANDROLOGY

Resistin, interleukin-6, tumor

necrosis factor-alpha, and human
semen parameters in the presence of
leukocytospermia, smoking habit,
and varicocele
Elena Moretti, Ph.D.,a Giulia Collodel, Ph.D.,a Lucia Mazzi, Ph.D.,a MariaStella Campagna, Ph.D.,b
Francesca Iacoponi, Ph.D.,c and Natale Figura, M.D., Ph.D.b
a
Department of Molecular and Developmental Medicine and b Department of Medical and Surgical Sciences and
Neurosciences, University of Siena, Siena; and c Department for Innovation in Biological, Agro-Food and Forest Systems,

della Tuscia, Viterbo, Italy
Universita

Objective: To explore the relationships between resistin, interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) and semen
parameters, sperm apoptosis, and necrosis in infertile patients and in control subjects with unknown reproductive potential with/
without smoking habits, leukocytospermia, and varicocele.
Design: Prospective study.
Setting: Sperm laboratory.
Patient(s): A total of 110 selected men.
Intervention(s): Family history, clinical/physical examination, ELISA determination (resistin, IL-6, TNF-a), semen analysis, annexin
V/propidium iodide assay.
Main Outcome Measure(s): Relationships among resistin, IL-6, and TNF-a and semen parameters in the presence of smoking habits,
varicocele, leukocytospermia, and in infertile subjects.
Result(s): Resistin level was higher in semen than in serum. Resistin semen levels showed negative correlations with sperm motility
and positive correlations with apoptotic, necrotic sperm and TNF-a and IL-6 levels. Resistin, TNF-a, and IL-6 levels were higher in
smokers compared with nonsmokers and in cases with leukocytospermia, in which an increase in necrotic sperm and a decrease in
the number of sperm with normal morphology and motility were observed. Cytokine levels were significantly higher in infertile
patients compared with control subjects with unknown reproductive potential. A total of 74.5% of infertile patients showed
leukocytospermia.
Conclusion(s): Semen resistin correlated with IL-6, TNF-a, and sperm quality; in cases of
leukocytospermia and smoking habits, resistin concentrations were increased, suggesting
that resistin may play a regulatory role in inflammation of the male reproductive system. Use your smartphone
(Fertil SterilÒ 2014;102:354–60. Ó2014 by American Society for Reproductive Medicine.) to scan this QR code
Key Words: Human sperm, IL-6, leukocytospermia, resistin, TNF-a and connect to the
discussion forum for
this article now.*
Discuss: You can discuss this article with its authors and with other ASRM members at http://
fertstertforum.com/morettie-resistin-il6-tnfa-leukocytospermia/ * Download a free QR code scanner by searching for “QR
scanner” in your smartphone’s app store or app marketplace.

R
esistin is a 12.5-kDa adipocyto- and secreted by adipocytes in mice and role in obesity-associated diabetes in
kine described by Steppan et al. capable of inducing insulin resistance men has been explored. However,
(1) as a new protein synthesized in rodents. Since then its potential studies revealed contradictory results
in humans and in rodents (2). Humans
Received January 29, 2014; revised and accepted April 10, 2014; published online May 13, 2014.
E.M. has nothing to disclose. G.C. has nothing to disclose. L.M. has nothing to disclose. M.C. has
and rodents share more than 50% sim-
nothing to disclose. F.I. has nothing to disclose. N.F. has nothing to disclose. ilarity in resistin amino acid sequence.
Reprint requests: Elena Moretti, Ph.D., Department of Molecular and Developmental Medicine, Uni- The expression of resistin by adipose
versity of Siena, Policlinico Le Scotte, Viale Bracci 14, 53100 Siena, Italy (E-mail: elena.moretti@
unisi.it). tissue varies significantly in different
species: in mice, resistin is almost
Fertility and Sterility® Vol. 102, No. 2, August 2014 0015-0282/$36.00
Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc.
exclusively expressed in white adipose
http://dx.doi.org/10.1016/j.fertnstert.2014.04.017 tissue, whereas low levels of resistin

354 VOL. 102 NO. 2 / AUGUST 2014


have been observed in mature human adipocytes (3). Recent
studies have reported that the expression of resistin in human
adipose tissue is mainly due to non-adipocyte resident in-
flammatory cells (4). In addition, analyses of the resistin
gene expression in a wide group of human tissues high-
lighted that the main source of resistin is made up of macro-
phages, peripheral blood mononuclear cells, and bone
marrow (2, 4–6).
The involvement of resistin in inflammation is indicated
by the fact that proinflammatory mediators, such as inter-
leukin (IL)-6, tumor necrosis factor-a (TNF-a), IL-1b, and
lipopolysaccharide, can increase resistin expression in pe-
ripheral mononuclear cells (3).
Many peptides, such as leptin, ghrelin, obestatin, and re-
sistin, are involved in the close relationship between repro-
ductive function and energy status (6, 7). Resistin, the topic
of the present study, was identified in the pituitary gland
and hypothalamus of mice; in addition, at the peripheral
level, resistin was detected by immunocytochemistry in
adult rat Leydig and Sertoli cells (8); the expression of this
peptide is controlled by gonadotropins, demonstrating that
resistin has a hormonal impact upon the testes (6).
Very few data are available on the presence and the im-
plications of resistin in human seminal plasma. The only pub-
lication dealing with resistin reported that it is correlated with
IL-6 and elastase in human semen (9). Recently Thomas et al.
(10) studied a panel of adipokines and detected increased re-
sistin concentrations in seminal plasma compared with serum
levels.
The aim of this study was to implement insights on the
relationship between resistin measured in seminal plasma
and serum and to study its possible associations with IL-6,
TNF-a, and semen parameters in the presence of different
conditions, such as smoking habits, leukocytospermia, and
varicocele. In addition, we compared the considered variables
between infertile patients and a group of control subjects with
unknown reproductive potential.
MATERIALS AND METHODS
Patients
From May 2013 through October 2013, we analyzed 152
consecutive semen samples from subjects (aged 22–49 years)
attending our center for semen analysis. One hundred ten pa-
tients were enrolled for this study according to the inclusion
criteria: non-azoospermic men with a normal karyotype eval-
uated by conventional cytogenetic analysis, body mass index
<25 kg/m2, and no history of diabetes, radiotherapy, chemo-
therapy, chronic illness, or medication. The subjects showed
normal concentrations of FSH, LH, and T evaluated in serum
by chemiluminescence using commercial kits (Beckman
Coulter Access for FSH, LH, and T).
The evaluation of the patients included information about
their medical history, lifestyle factors such as the use of ciga-
rettes, drugs, and alcohol, clinical and physical examinations,
and scrotal Eco-color Doppler to explore the possible presence
of varicocele.
Semen samples were obtained from patients with fertility
problems and from men who wanted to be tested for fertility.
VOL. 102 NO. 2 / AUGUST 2014
Fertility and Sterility®
Infertile patients were individuals who did not obtain preg-
nancy after 2 years of unprotected sexual intercourse (n ¼
47); the others were individuals who were not looking for
an outcome of pregnancy but came to our center to control
their semen parameters (n ¼ 63).
Considering clinical history, physical examination, and
routine semen analysis, we identified groups of patients
showing dichotomous characteristics—smoking habits (>10
cigarette per day for more than 5 years), leukocytospermia
(>1  106 leukocytes/mL of semen) (11), a past history of
varicocele (patients who had varicocele in the past; all pa-
tients had correction of varicocele; by physical examination
and scrotal Eco-color Doppler all the subjects showed nega-
tive results at enrollment), and the presence/absence of vari-
cocele—to determine whether these situations could be related
to resistin levels measured in semen. All patients provided
informed written consent before the inclusion on this study,
which was approved by the Ethics Committee of Azienda
Ospedaliera Universitaria Senese.
Semen Analysis
Semen samples were collected by masturbation after 3 to
4 days of sexual abstinence and analyzed after liquefaction
for 30 minutes at 37 C. Volume, pH, sperm concentration,
motility, and morphology (Papanicolaou test modified for
spermatozoa) were evaluated according to 2010 World Health
Organization guidelines (11). Leukocytes were identified by
peroxidase stain; a concentration >1  106 cells/mL was
considered out of range (11) and identified as
leukocytospermia.
Detection of Membrane Phosphatidylserine
Externalization and Membrane Integrity Using the
Annexin V/Propidium Iodide Assay
The detection of phosphatidylserine (PS) externalization and
membrane integrity was performed by Vybrant apoptosis
assay (Invitrogen) based on fluorescein isothiocyanate–an-
nexin V (AnV, green fluorescence) and propidium iodide
(PI, red fluorescence). These compounds are able to label
dead cells, differentiating apoptosis and necrosis. The translo-
cation of PS, recognized by annexin V protein, from the inner
to the outer plasma membrane layer, is a critical step in
apoptosis; PI enters inside necrotic cells with broken
membranes.
Aliquots of 110 semen samples were washed with PBS,
centrifuged, suspended in annexin-binding buffer to obtain
a cell density of approximately 1  106 sperm/mL, and finally
treated according to the manufacturer's instructions. A drop
of sperm suspension was smeared on glass slide and then
mounted in glycerol containing 5% n-Propyl gallate. Obser-
vations and photographs were made with a Leitz Aristoplan
light microscope (Leica) equipped with a fluorescence appa-
ratus. A minimum of 300 sperm was scored for each sample,
identifying intact cells (unstained: AnV negative, PI nega-
tive), apoptotic cells (green: AnV positive, PI negative), and
necrotic sperm (red: AnV negative, PI positive). Sperm with
355
ORIGINAL ARTICLE: ANDROLOGY
both green and red signals were considered necrotic because
they had a broken plasma membrane.
Resistin, IL-6, and TNF-a Measurements
Resistin, IL-6, and TNF-a were assayed in semen samples of
110 individuals; resistin levels were also measured in 36 blood
serum samples of homologous subjects.
For each semen sample, 500 mL of the whole semen were
recovered 1 hour after collection and fractioned by centrifu-
gation (1,500 rpm for 15 minutes). The supernatant,
composed of seminal plasma without spermatozoa, was
immediately stored at 80 C until analyses were performed.
Blood samples were collected after 12 hours of fasting be-
tween 9:00 and 10:30 AM from a cubital vein and were drawn
in color-coded Vacutainer tubes (BD). Blood was allowed to
clot for approximately 60 minutes, separated by centrifuga-
tion at 4 C, and stored at 80 C until resistin and cytokine
levels were assayed.
Resistin levels were determined by ELISA using the Resis-
tin (Human) ELISA kit (Phoenix Pharmaceutical). Following
the manufacturer's instructions, the results were expressed
in nanograms per milliliter.
Interleukin-6 and TNF-a levels were determined by
ELISA (Human IL-6 BMS213/2CE BMS213/2TENCE and
TNF-a BMS223/4CE BMS223/4TENCE; Bender MedSystems),
and the results were expressed in picograms per milliliter as
reported in the manufacturer's instructions.
Statistical Analysis
Data were reported as mean and SD, and the normality of dis-
tribution was tested using the Shapiro-Wilk test. The Levene
test was performed to assess the homogeneity of variances.
The paired t test was used for comparisons between semen
and serum levels of resistin. The Pearson correlation coeffi-
cient was calculated for bivariate correlation analysis. Chi-
square tests were used to highlight the possible associations
between the dichotomous variables (smoking habits, leukocy-
tospermia, varicocele, and history of corrected varicocele). We
used the Mann-Whitney U test to compare the groups derived
from the stratified population. The same test was also applied
to compare the variables between control subjects with un-
known reproductive potential (group A) and the group of
infertile men (group B). A P value of < .05 (two-tailed) was
considered significant. Statistical analysis was performed us-
ing the SPSS 16 statistical package.
RESULTS
The characteristics of the 110 patients are shown in Table 1.
The mean sperm concentrations resulted between the 25th
and 50th centiles, the mean progressive motility percentages
fell between the 5th and 10th centiles, and the mean percent-
ages of sperm with normal morphology were between the
50th and 75th centiles (11). The mean (SD) percentages of
sperm with apoptosis and necrosis were respectively 10%
(6%) and 24% (13%).
The presence of resistin was observed in all analyzed
semen and serum samples. Because resistin was also
356
TABLE 1
Age and considered semen variables in 110 individuals.
Variable n Mean (SD)
Age, y 110 33.50 (8.13)
Semen parameters 110
Volume, mL 3.3 (1.4)
Sperm, mL  106 69 (42)
Progressive motility, % 34 (14)
Normal morphology, % 24 (8)
Apoptosis, % 10 (6)
Necrosis, % 24 (13)
IL-6, pg/mL 19 (34)
TNF-a, pg/mL 48.2 (70.9)
Resistin levels
Serum, ng/mL 36 1.08 (0.58)
Semen, ng/mL 110 2.4 (2)a
Note: IL-6, TNF-a, and resistin levels were determined in all semen samples. Resistin concen-
trations were also measured in the serum of 36 men.
a
The mean (SD) values of resistin referring to the 36 individuals in whom the semen vs. serum
comparison was performed was 2.75 (1.9) ng/mL.
Moretti. Resistin, IL-6, TNF-a, and semen parameters. Fertil Steril 2014.
measured in the serum of 36 individuals, the comparison be-
tween serum and semen resistin was performed only in these
cases. No significant correlation was observed between resis-
tin levels measured in serum and semen samples (r ¼ 0.002;
P¼ .991). The mean level of resistin in semen (2.75 ng/mL,
SD 1.9) was significantly higher (P< .001) than that measured
in serum samples (1.08 ng/mL, SD 0.58) of the 36 homologous
individuals in whom this comparison was performed.
The correlations between the variables considered in this
study are shown in Table 2. Concerning sperm parameters,
significant positive correlations were observed between
sperm motility and sperm concentration (r ¼ 0.39; P< .001)
and between percentages of sperm with normal morphology
and sperm concentrations (r ¼ 0.59; P< .001). The percentage
of apoptotic sperm showed significant negative correlations
with sperm concentration (r ¼ 0.24; P< .01), motility (r ¼
0.43; P< .001), and percentage of sperm with normal
morphology (r ¼ 0.33; P< .001). The percentage of necrotic
sperm was negatively correlated with sperm concentration
(r ¼ 0.37; P< .001), motility (r ¼ 0.74; P< .001), and per-
centage of sperm with normal morphology (r ¼ 0.31;
P< .001); the percentage of necrotic sperm correlated posi-
tively with apoptosis (r ¼ 0.33; P< .001).
Interesting relationships were observed when we compared
levels of TNF-a, IL-6, and resistin. The TNF-a and IL-6 levels
correlated positively with the percentages of necrotic sperm
(respectively, r ¼ 0.42; P< .001 and r ¼ 0.51; P< .001) and
negatively with motility (respectively, r ¼ 0.27; P< .01 and
r ¼ 0.32; P< .001). In addition, TNF-a correlated positively
with IL-6 (r ¼ 0.71; P< .001). Resistin levels showed a signifi-
cant negative correlation with the percentage of sperm motility
(r ¼ 0.39; P< .001) and positive correlations with the percent-
ages of apoptotic (r ¼ 0.21; P< .05) and necrotic (r ¼ 0.63;
P< .001) sperm and with TNF-a (r ¼ 0.42; P< .001) and IL-6
(r ¼ 0.59; P< .001) levels.
The use of specific statistics enabled us to consider the
dichotomous variables as independent, and comparisons
were performed for all the considered characteristics in the
VOL. 102 NO. 2 / AUGUST 2014
 Fertility and Sterility®

following groups: [1] smokers–nonsmokers, [2] leukocyto-

serum, ng/mL
spermia–no leukocytospermia, [3] history of corrected varico-

Resistin

1.00
cele–no history of corrected varicocele, and [4] varicocele–no
varicocele (Table 3). In group 1, the mean levels of TNF-a, IL-
6, and resistin were significantly higher in smokers (P< .05)
compared with those observed in nonsmokers. In group 2,
semen, ng/mL

the mean percentages of sperm motility and sperm with

Resistin

1.00
0.00
normal morphology were significantly decreased in patients
with leukocytospermia compared with those detected in pa-
tients without leukocytospermia (respectively, P< .001 and
P< .01). In the same group, in the presence of leukocytosper-
IL-6, pg/mL

0.59***
mia, we observed a significant increase in necrotic sperm per-
1.00 centages and TNF-a, IL-6, and semen resistin levels (P< .001)
0.16
(Table 3). In group 3, sperm concentrations in men with a his-
tory of varicocele were significantly lower (P< .05) than those
TNF-a, pg/mL

detected in men without a history of varicocele. In group 4,

0.71***
0.42***

the percentages of motile spermatozoa were significantly

1.00

0.17

decreased (P< .01), and the percentage of necrotic sperm

increased (P< .01) in subjects with varicocele (Table 3). It is
noteworthy that in this group the levels of TNF-a, IL-6, and
Necrosis, %

resistin were increased, although in a nonsignificant manner

0.42***
0.51***
0.63***

(Table 3).
1.00

0.01

No significant differences in the concentrations of TNF-a,

IL-6, and semen resistin were observed in group 3.
To investigate the behavior of resistin in infertile patients,
Apoptosis, %

0.33***

we divided the study participants into two groups: group A

0.21*
1.00

0.02
0.13

0.27

was composed of control subjects with unknown reproductive

potential, and group B was composed of infertile men. The re-
sults of the comparison are reported in Table 4. Semen param-
morphology, %

eters were significantly altered (P< .001), and the levels of

0.33***
0.31***
Normal

cytokines increased (P< .001) in group B vs. group A

1.00

0.07
0.08
0.11
0.07

(Table 4). The frequency of considered dichotomous condi-

tions in the two groups was as follows: 30.1% of smokers in
Bivariate correlations between all considered variables (Pearson correlation coefficient).

group A and 42.5% in group B; 38.1% of subjects with leuko-

cytospermia in group A and 74.5% of patients in group B;
Motility, %

0.43***
0.74***

0.32***
0.39***
0.27**

25.39% of individuals with varicocele in group A and

1.00
0.44

0.02

38.29% in group B; 20.6% of men with history of corrected

varicocele in group A and 23.4% in group B.
Sperm/mL, 3106

0.39***
0.59***

0.37***

DISCUSSION
0.24**
1.00

0.18
0.13
0.15
0.03

Evidence of the role of resistin in inflammation is available in

the literature (2–4, 12). Resistin is strictly related to the
Moretti. Resistin, IL-6, TNF-a, and semen parameters. Fertil Steril 2014.

proinflammatory cytokine pathway, and it can target

several human cells, enhancing inflammatory and
volume, mL
Semen

autoimmune processes (3).

1.00
0.05
0.19
0.04
0.11
0.03
0.10
0.05
0.01
0.10

In the reproductive system resistin was found to be ex-

pressed in rat Leydig and Sertoli cells (8) and also in macro-
phages, which represent a major population among immune
1.00
0.15
0.00
0.07
0.08
0.06
0.12
0.05
0.07
0.06
0.19
Age, y

cells in the testicular interstitial region; in addition, cytokines,

including IL-6 and TNF-a, are produced in the testis, where
they regulate testicular functions under physiologic condi-
*P< .05; **P< .01; ***P< .001.
Normal morphology, %

Resistin semen, ng/mL

Resistin serum, ng/mL

tions (13). During inflammation, however, the levels of cyto-

Semen volume, mL

kines and reactive oxygen species increase, thus impairing the

Sperm/mL, 106

spermatogenetic process (13–15). The role of resistin is

Apoptosis, %

TNF-a, pg/mL
Necrosis, %
TABLE 2

Motility, %

IL-6, pg/mL

becoming clearer, but new ideas on resistin receptors are

Parameter

very recent. Studies identified an isoform of decorin, a

Age, y

component of extracellular matrix, as a functional resistin

receptor in an adipocyte progenitor (16, 17). Decorin is

VOL. 102 NO. 2 / AUGUST 2014 357

ORIGINAL ARTICLE: ANDROLOGY

present in human testis, and increased decorin production,

Mean (SD) of all considered variables reported in group 1 (smokers vs. nonsmokers), group 2 (leukocytospermia vs. no leukocytospermia), group 3 (history of corrected varicocele vs. no history of

7.02

0.62
1.5

116.0

2.0
SD
TNF-a dependent, is associated with impaired testicular

37
12

12

47
9
6
Yes (34)
function (18). A further binding site for resistin was
Group 4: varicocele recognized as Toll-like receptor 4 detected in hypothalamus

30.41

1.09
Mean

28**

29**
64.6
3.3

2.9
(19); it is noteworthy that such a receptor has also been found

61

23
11

27
in human sperm (20), in rat epididymal epithelium and sperm
(21), and in the testis and epididymis of roosters (22).
8.25

0.58
1.3

1.9
34.9
SD
To the best of our knowledge, however, specific studies

8
6
44
14

13

27
No (76)

explaining the mechanism of action of resistin in the male hu-

man reproductive system are still lacking.

1.07
34.91
Mean

3.4

2.2
40.9
The present study aimed at exploring the presence of re-
73
36
25

22

15
9 sistin in human semen and to investigate its relationship with
IL-6, TNF-a, and sperm parameters; in addition, we have re-
6.20

0.68
1.4

137.6

2.0
ported the behavior of resistin and cytokines in the presence
SD

6
7
38
14

12

42
Group 3: history of corrected varicocele

of particular conditions affecting semen quality: smoking

Yes (24)

habits, leukocytospermia, varicocele, and history of corrected

varicocele. Finally, we performed a comparison between vari-
31.63

1.16
Mean

ables in a group of infertile patients and a group of control

3.6

2.1
62.2
52*
34
24
10
23

16

subjects with unknown reproductive potential.

We observed a relationship between the levels of resistin
and IL-6 in seminal plasma of our patients. A similar finding
8.55

0.54

has also been reported by Kratzsch et al. (9).

1.4

2.0
35.4
SD

8
6
42
14

13

32

The significant correlation between resistin and TNF-a

No (86)

concentrations in semen samples is novel; the relationship

between the amounts of IL-6 and TNF-a in the semen con-
1.04
34.04
Mean

firms the existence of a link between these factors, which re-

3.3

2.5
44.3
74
34
24
10
24

20

inforces the hypothesis that resistin is involved in the

cytokine network during inflammation.
Our results are different from those reported by the first
8.27

0.64
92.5
1.5

1.9
SD

publication in this field (9), which described a relationship be-

38
12

12

43
8
6
Yes (59)
Group 2: leukocytospermia

tween resistin and IL-6 but not with the semen quality. We
3.1***
65.1***

demonstrated a negative influence of resistin, IL-6, and

Mean

29***

29***

29***
33.25

1.05
23**
3.5

TNF-a upon sperm motility and positive correlations between

63

10

resistin (and analyzed cytokines) and indices of sperm death:

necrosis and apoptosis. The negative effect of increased levels
8.03

0.50
17.6
1.2

1.7
SD

of cytokines on sperm parameters has been reported by other

45
14

12

14
8
6
No (51)

groups (23–27), and the discrepancy between the results of

this study and those obtained by Kratzsch et al. (9) could be
1.12
33.80
Mean

3.2

1.5
28.6

due to the selection of patients. Our observation is also

76
39
26

19
9

supported by in vitro studies reporting that IL-6 and TNF-a

corrected varicocele), and group 4 (varicocele vs. no varicocele).

significantly reduced sperm progressive motility in a dose-

6.64

0.52
1.4

1.9
30.4
SD

dependent manner (28). In addition, bivariate correlation

46
13

12

33
9
6
Yes (39)

analysis enabled us to highlight significant positive relation-

Group 1: smokers

32.74

49.3*

2.9*
1.08
Mean

Moretti. Resistin, IL-6, TNF-a, and semen parameters. Fertil Steril 2014.

ships between some sperm characteristics, such as concentra-

3.4

23*
78
33
25
10
25

tion, motility, and normal morphology, and significant

negative correlations between these sperm parameters and
8.87

0.61

viability scores.
1.4

1.9
85.6
SD

8
6
39
14

14

35
No (71)

We also demonstrated that resistin is present in seminal

plasma at increased concentrations compared with the serum
1.07
33.93
Mean

3.3

2.1
47.6

levels of homologous patients, but this issue is still debated in

64
34
24

24

17
9

the literature (9, 10). No linear relationship between semen

and serum resistin was observed in our patient population.
*P< .05; **P< .01; ***P< .001.
Normal morphology, %

Resistin semen, ng/mL

Resistin serum, ng/mL

Our results argue against a hypothetical diffusion of resistin

Semen volume, mL

from serum to seminal plasma, and they suggest that

Sperm/mL, 106

resistin can be produced directly in the testis, accessory

Apoptosis, %

TNF-a, pg/mL
Necrosis, %
TABLE 3

Motility, %

IL-6, pg/mL

glands, and macrophages residing in the male reproductive

Parameter

tract. Interesting results were obtained by grouping the

Age, y

patients according to the presence of particular conditions

that may affect the semen quality: smoking habits,

358 VOL. 102 NO. 2 / AUGUST 2014

 Fertility and Sterility®

concentrations of IL-6 and TNF-a, chemokines well known

TABLE 4
to exert a negative effect on sperm quality (2, 28, 35, 36).
Comparison between the considered variables in group A, composed
We hypothesize that the presence of leukocytes in semen,
of control subjects with unknown reproductive potential, and group increasing levels of resistin and other cytokines and
B, composed of infertile men. decreasing semen quality, could indicate a typical
Median (IQR P inflammation profile.
Variable Group 75th–25th centile) value It is noteworthy that in the group of individuals with vari-
Age, y A 31 (11.25) .089 cocele the sperm motility, as expected, was reduced (37);
B 35 (8) concomitantly, the sperm necrosis and the levels of analyzed
Semen volume, mL A 3 (1.7) .150 chemokines were increased. Such observations suggest that
B 3.5 (2.3)
Sperm/mL, 10 6
A 77 (42.75) < .001 varicocele might induce an inflammatory effect that could
B 49 (63.5) play a detrimental role in spermatogenesis (14); such a hy-
Motility, % A 44.5 (17.5) < .001 pothesis is supported by the demonstration that the presence
B 23 (10)
Normal morphology, % A 26 (10.5) < .001
of this pathology increases the levels of reactive oxygen spe-
B 20 (11) cies and reduces the total antioxidant capacity (38).
Apoptosis, % A 7 (5.75) < .001 It is noteworthy that resistin, IL-6, and TNF-a concentra-
B 12 (7) tions seemed to be significantly higher in a group of infertile
Necrosis, % A 15.5 (14.5) < .001
B 35.00 (11) patients compared with those observed in control subjects
TNF-a, pg/mL A 25.2 (12.25) < .001 with unknown reproductive potential. These results could be
B 45.6 (50.2) of particular interest and deserve attention in the field of
IL-6, pg/mL A 3.55 (7.55) < .001 male infertility; however, other studies involving groups of
B 14.1 (31.4)
Resistin semen, ng/mL A 0.6 (1.85) < .001 infertile patients and proven fertile controls are mandatory
B 4 (2.9) because our comparison group was composed of individuals
Note: P values obtained with Mann-Whitney U tests. IQR ¼ interquartile range. who were not trying to have a baby yet. In any event, these
Moretti. Resistin, IL-6, TNF-a, and semen parameters. Fertil Steril 2014. data confirmed the relationship between increased levels of
analyzed chemokines and leukocytospermia: 74.5% of infer-
leukocytospermia, and the presence of varicocele and/or tile patients showed this condition (vs. 38.1% of men occurred
history of corrected varicocele. We observed a significant for control).
increase in resistin, IL-6, and TNF-a concentrations in semen In conclusion, resistin levels in seminal plasma are corre-
samples of cigarette smokers compared with nonsmokers. lated with other inflammatory cytokines and sperm quality;
Current dogma suggests that the persistent particulate/oxida- in semen samples with leukocytospermia and in those ob-
tive burden caused by smoking can generate secondary medi- tained from smokers, concentrations of resistin were
ators, such as cytokines, growth factors, and proteases, which increased. These observations may support a role of resistin
may induce inflammatory diseases mainly involving the pul- as a potential regulator of inflammation in the male reproduc-
monary district (29). To the best of our knowledge, studies on tive system. Our preliminary results might suggest a putative
cytokines detection in the semen specimens of cigarette role of resistin as a marker of inflammation, even if other
smokers are lacking; thus, our observation could encourage studies are necessary to confirm these observations; in partic-
other researches to clarify the mechanisms through which ular, it would be interesting to measure the seminal levels of
smoking may reduce semen quality. resistin and reactive oxygen species, which is the gold stan-
The observations that peripheral blood mononuclear dard in the diagnosis of inflammation.
cells, macrophages, and neutrophils express resistin involved
in the inflammatory pathway (3, 4, 12, 30) prompted us to
explore its involvement in leukocytospermia. In our patients REFERENCES
the presence of leukocytospermia was concomitant with a 1. Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, et al. The
reduction of sperm motility and percentages of sperm with hormone resistin links obesity to diabetes. Nature 2001;409:307–12.
normal forms and with an increase in necrosis. Other 2. Stofkova A. Resistin and visfatin: regulators of insulin sensitivity, inflamma-
authors observed that leukocytospermia could exert a tion and immunity. Endocr Regul 2010;44:25–36.
3. Filkova M, Haluzík M, Gay S, Senolt L. The role of resistin as a regulator of
negative effect on semen quality (31, 32); however,
inflammation: implications for various human pathologies. Clin Immunol
emerging evidence indicates that moderate
2009;133:157–70.
leukocytospermia seems to be physiologic (33). In semen, 4. Schwartz DR, Lazar MA. Human resistin: found in translation from mouse to
leukocytes are mainly represented by polymorphonuclear man. Trends Endocrinol Metab 2011;22:259–65.
granulocytes that can originate from two regions: the 5. Kaser S, Kaser A, Sandhofer A, Ebenbichler CF, Tilg H, Patsch JR. Resistin
epididymis, in which a small number of activated messenger-RNA expression is increased by proinflammatory cytokines
leukocytes may play a positive role in sperm quality, and in vitro. Biochem Biophys Res Commun 2003;309:286–90.
6. Michalakis K, Mintziori G, Kaprara A, Tarlatzis BC, Goulis DG. The complex
the prostate, in which a large number of polymorphs may
interaction between obesity, metabolic syndrome and reproductive axis: a
reflect prostatic disorders (34). We demonstrated, for the narrative review. Metabolism 2013;62:457–78.
first time, that human semen samples with 7. Budak E, Fernandez Sanchez M, Bellver J, Cervero A, Simon C, Pellicer A. In-
leukocytospermia showed increased levels of resistin; the teractions of the hormones leptin, ghrelin, adiponectin, resistin, and PYY3-
highest levels of resistin were concomitant with enhanced 36 with the reproductive system. Fertil Steril 2006;85:1563–81.

VOL. 102 NO. 2 / AUGUST 2014 359

ORIGINAL ARTICLE: ANDROLOGY
8. Nogueiras R, Barreiro ML, Caminos JE, Gaytan F, Suominen JS, Navarro VM,
et al. Novel expression of resistin in rat testis: functional role and regulation
by nutritional status and hormonal factors. J Cell Sci 2004;117:3247–57.
9. Kratzsch J, Paasch U, Grunewald S, Mueller MA, Thiery J, Glander HJ. Resis-
tin correlates with elastase and interleukin-6 in human seminal plasma. Re-
prod Biomed Online 2008;16:283–8.
10. Thomas S, Kratzsch D, Schaab M, Scholz M, Grunewald S, Thiery J, et al.
Seminal plasma adipokine levels are correlated with functional characteris-
tics of spermatozoa. Fertil Steril 2013;99:1256–63.
11. World Health Organization. WHO laboratory manual for the examination
and processing of human semen. 5th ed. Geneva: WHO Press; 2010.
12. Walcher D, Hess K, Berger R, Aleksic M, Heinz P, Bach H, et al. Resistin: a
newly identified chemokine for human CD4-positive lymphocytes. Cardio-
vasc Res 2010;85:167–74.
13. Li N, Wang T, Han D. Structural, cellular and molecular aspects of immune
privilege in the testis. Front Immunol 2012;3:152.
14. Moretti E, Cosci I, Spreafico A, Serchi T, Cuppone AM, Collodel G. Semen
characteristics and inflammatory mediators in infertile men with different
clinical diagnoses. Int J Androl 2009;32:637–46.
15. Sarkar O, Bahrainwala J, Chandrasekaran S, Kothari S, Mathur PP, Agarwal A.
Impact of inflammation on male fertility. Front Biosci 2011;3:89–95.
16. Daquinag AC, Zhang Y, Amaya-Manzanares F, Simmons PJ, Kolonin MG. An
isoform of decorin is a resistin receptor on the surface of adipose progenitor
cells. Cell Stem Cell 2011;9:74–86.
17. Onuma H, Tabara Y, Kawamura R, Ohashi J, Nishida W, Takata Y, et al.
Plasma resistin is associated with single nucleotide polymorphisms of a
possible resistin receptor, the decorin gene, in the general Japanese popu-
lation. Diabetes 2013;62:649–52.
18. Adam M, Urbanski HF, Garyfallou VT, Welsch U, Ko €hn FM, Ullrich
Schwarzer J, et al. High levels of the extracellular matrix proteoglycan de-
corin are associated with inhibition of testicular function. Int J Androl
2012;35:550–61.
19. Benomar Y, Gertler A, De Lacy P, Crepin D, Ould Hamouda H, Riffault L, et al.
Central resistin overexposure induces insulin resistance through Toll-like re-
ceptor 4. Diabetes 2013;62:102–14.
20. Fujita Y, Mihara T, Okazaki T, Shitanaka M, Kushino R, Ikeda C, et al. Toll-like
receptors (TLR) 2 and 4 on human sperm recognize bacterial endotoxins and
mediate apoptosis. Hum Reprod 2011;26:2799–806.
21. Cao D, Li Y, Yang R, Wang Y, Zhou Y, Diao H, et al. Lipopolysaccharide-
induced epididymitis disrupts epididymal beta-defensin expression and in-
hibits sperm motility in rats. Biol Reprod 2010;83:1064–70.
22. Zhang M, Nii T, Isobe N, Yoshimura Y. Expression of Toll-like receptors and
effects of lipopolysaccharide on the expression of proinflammatory cyto-
kines and chemokine in the testis and epididymis of roosters. Poult Sci
2012;91:1997–2003.
23. Eggert-Kruse W, Boit R, Rohr G, Aufenanger J, Hund M, Strowitzki T. Rela-
tionship of seminal plasma interleukin (IL)-8 and IL-6 with semen quality.
Hum Reprod 2001;16:517–28.
360
24. Eggert-Kruse W, Kiefer I, Beck C, Demirakca T, Strowitzki T. Role for tumor
necrosis factor alpha (TNF-alpha) and interleukin 1-beta (IL-1beta) determi-
nation in seminal plasma during infertility investigation. Fertil Steril 2007;87:
810–23.
25. Sanocka D, Jedrzejczak P, Szuma1a-Kaekol A, Fraczek M, Kurpisz M. Male
genital tract inflammation: the role of selected interleukins in regulation
of pro-oxidant and antioxidant enzymatic substances in seminal plasma. J
Androl 2003;24:448–55.
26. Fraczek M, Kurpisz M. Inflammatory mediators exert toxic effects of oxida-
tive stress on human spermatozoa. J Androl 2007;28:325–33.
27. Carrasquel G, Camejo MI, Michelangeli F, Ruiz MC. Effect of tumor necrosis
factor-a on the intracellular Ca2þ homeostasis in human sperm. Am J Re-
prod Immunol 2013;70:153–61.
28. Lampiao F, du Plessis SS. TNF-alpha and IL-6 affect human sperm function
by elevating nitric oxide production. Reprod Biomed Online 2008;17:
628–31.
29. Rastrick JM, Stevenson CS, Eltom S, Grace M, Davies M, Kilty I, et al. Ciga-
rette smoke induced airway inflammation is independent of NF-kB signal-
ling. PLoS One 2013;8:e54128.
30. Kunnari AM, Savolainen ER, Ukkola OH, Kes€aniemi YA, Jokela MA. The
expression of human resistin in different leucocyte lineages is modulated
by LPS and TNFalpha. Regul Pept 2009;157:57–63.
31. Mahfouz R, Sharma R, Thiyagarajan A, Kale V, Gupta S, Sabanegh E, et al.
Semen characteristics and sperm DNA fragmentation in infertile men with
low and high levels of seminal reactive oxygen species. Fertil Steril 2010;
94:2141–6.
32. Aghazarian A, Stancik I, Pfl€ uger H, Lackner J. Influence of pathogens and
moderate leukocytes on seminal interleukin (IL)-6, IL-8, and sperm parame-
ters. Int Urol Nephrol 2013;45:359–65.
33. Barraud-Lange V, Pont JC, Ziyyat A, Pocate K, Sifer C, Cedrin-Durnerin I,
et al. Seminal leukocytes are Good Samaritans for spermatozoa. Fertil Steril
2011;96:1315–9.
34. Ziyyat A, Barraud-Lange V, Sifer C, Ducot B, Wolf JP, Soufir JC. Paradox-
ical increase of sperm motility and seminal carnitine associated with
moderate leukocytospermia in infertile patients. Fertil Steril 2008;90:
2257–63.
35. Perdichizzi A, Nicoletti F, La Vignera S, Barone N, D'Agata R, Vicari E, et al.
Effects of tumour necrosis factor-alpha on human sperm motility and
apoptosis. J Clin Immunol 2007;27:152–62.
36. Martínez-Prado E, Camejo Bermu dez MI. Expression of IL-6, IL-8, TNF-alpha,
IL-10, HSP-60, anti-HSP-60 antibodies, and anti-sperm antibodies, in semen
of men with leukocytes and/or bacteria. Am J Reprod Immunol 2010;63:
233–43.
37. Will MA, Swain J, Fode M, Sonksen J, Christman GM, Ohl D. The great
debate: varicocele treatment and impact on fertility. Fertil Steril 2011;95:
841–52.
38. Tawadrous GA, Aziz AA, Mostafa T. Seminal soluble fas relationship with
oxidative stress in infertile men with varicocele. Urology 2013;82:820–3.
VOL. 102 NO. 2 / AUGUST 2014