Human Reproduction Vol.19, No.8 pp. 1821–1825, 2004 DOI: 10.

1093/humrep/deh324
Advance Access publication May 27, 2004

A combination of interleukin-6 and its soluble receptor

impairs sperm motility: implications in infertility
associated with endometriosis

Souichi Yoshida1, Tasuku Harada, Tomio Iwabe, Fuminori Taniguchi, Masahiro Mitsunari,
Nobuhiro Yamauchi, Imari Deura, Sayako Horie and Naoki Terakawa
Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683-8504, Japan
1
To whom correspondence should be addressed. E-mail: souichi@grape.med.tottri-u.ac.jp

BACKGROUND: We previously reported that the level of interleukin (IL)-6 is increased in the peritoneal fluid
of women with endometriosis. This study was undertaken to assess the effects of IL-6 and soluble IL-6 receptor

Downloaded from http://humrep.oxfordjournals.org/ by guest on November 15, 2015

(sIL-6R) on in vitro sperm motility. METHODS: Sperm (n 5 20) were cultured with IL-6 or sIL-6R, or with a
combination of both. After 24 h cultures, sperm motility was evaluated using a computer-assisted semen analysis
system. Gene and protein expressions of IL-6, IL-6 receptor (IL-6R), and glycoprotein 130 (gp130) were examined
in sperm by RT – PCR analysis and western blot analysis. RESULTS: Addition of IL-6 or sIL-6R individually to
the culture media had no affect on sperm motion. However, adding a combination of IL-6 and sIL-6R dose-
dependently reduced the percentage of motile and rapidly moving sperm. Adding anti-IL-6R antibody abolished
these adverse effects. Sperm expressed the gp130 gene and protein, but not IL-6 or IL-6R. CONCLUSIONS: A
combination of IL-6 and sIL-6R may be associated with gp130 expressed in the sperm and reduce sperm motility.
IL-6 and sIL-6R may contribute to the pathogenesis of endometriosis-associated infertility.

Key words: glycoprotein 130/interleukin-6/soluble interleukin-6 receptor/sperm motility/peritoneal fluid

Introduction Fallopian tube (Lyons et al., 2002). Since the PF of infertile

Endometriosis, characterized by the presence and growth of
endometrial cells outside the uterus that cause pelvic pain, is
often associated with infertility. Infertility can occur even in
the early stages of endometriosis when adhesions or anatom-
ical distortion are not yet present. Although the pathogenesis
of endometriosis-associated infertility is poorly understood, a
number of authors have suggested possible causes, such as
tubal occlusion due to adhesions (Haney, 1991), a defect in
granulosa cell steroidgenesis (Harlow et al., 1996), occur-
rence of luteinized unruptured follicles (Mio et al., 1992),
an inadequate luteal phase (Hirschowitz et al., 1987), and
association of auto-antibodies (Fernandez-Shaw et al., 1993).
Moreover, accumulated data suggest that the peritoneal
microenvironment may contribute to endometriosis-associ-
ated infertility and to the pathogenesis of endometriosis
(Harada et al., 2001).
The volume of peritoneal fluid (PF) is significantly elev-
ated in infertile women with endometriosis compared to those
without endometriosis (Harada et al., 1997a). PF is reported
to be associated with infertility in several ways, e.g. inhibiting
sperm motion (Drudy et al., 1994; Aeby et al., 1996), sperm
acrosome reaction (Arumugam, 1994), mouse embryo growth
(Prough et al., 1990), sperm binding to the zona pellucida
(Coddington et al., 1992), and ciliary action in the human
Human Reproduction vol. 19 no. 8 q European Society of Human Reproduction and Embryology 2004; all rights reserved
women with endometriosis contains abundant cytokines and
growth factors, these factors may exert those detrimental
effects on the reproductive process and possibly contribute to
endometriosis-associated infertility (Harada et al., 2001).
Interleukin-6 (IL-6), a pleiotropic cytokine produced by a
variety of cell types, plays a pivotal role as a mediator of
numerous physiological and pathogenic processes (Tagoh
et al., 1989). It has also been suggested that IL-6 has import-
ant functions in reproductive physiology, including the regu-
lation of ovarian steroid production, folliculogenesis, and
early events related to implantation (Gorospe et al., 1992;
Ray et al., 1997). IL-6 exerts these effects to bind cognate
membrane receptor complexes, consisting of an 80 kDa
ligand binding subunit [glycoprotein (gp) 80: interleukin-6
receptor; IL-6R] and a 130 kDa signal transducing protein
(gp130). IL-6R binds IL-6 with low affinity and must associ-
ate with gp130 for high affinity binding and signal transduc-
tion to occur (Jacobs et al., 1992). A 55 kDa soluble form of
IL-6R (soluble IL-6 receptor; sIL-6R), which is proteolyti-
cally cleaved from IL-6R, exists in body fluid. It can enhance
the bioactivity of IL-6 by direct binding of the IL-6/sIL-6R
complex to gp130 (Rose-John et al., 1994).
We previously reported that levels of IL-6 in PF were
significantly higher in infertile women with endometriosis
1821
S.Yoshida et al.
and that its soluble receptor also exists in PF (Harada et al.,
1997a). Although the main source of IL-6 had been con-
sidered to be macrophages, our previous study demonstrated
that IL-6 is produced in endometriotic stromal cells at levels
similar to that in macrophages (Tsudo et al., 2000).
Accordingly, we hypothesize that IL-6 and sIL-6R may be
responsible for the reported adverse effects on reproductive
processes of PF from patients with endometriosis. In this
study, we focused on the effects of IL-6 and sIL-6R on
in vitro sperm motility. In addition, we also examined the
expressions of IL-6, IL-6R and gp130 in sperm.
Materials and methods
Semen samples and washing of sperm
Semen samples were obtained from 20 normozoospermic fertile vol-
unteers who met the diagnostic criteria of the World Health Organ-
ization (WHO) standards (1992) and abstained from coitus for
3 days. Informed consent was obtained from all subjects. The semen
samples were processed by the Percoll (Sigma Co., USA) gradient
technique (three layers: 40, 70 and 90%) to separate seminal plasma
and to remove cell contamination. The supernatant was removed,
and the pellet was resuspended in 2 ml human tubal fluid medium
(HTF; Irvine Scientific, USA). The tube was centrifuged at 500 g for
10 min. A swim-up migration was then performed by placing a layer
of 1 ml serum-free fresh medium over the sperm pellet. The tube
was then incubated for 45 min at 378C under an atmosphere of 5%
CO2 in air. The supernatant was gently aspirated and sperm concen-
tration and motility evaluated. A part of the supernatant was centri-
fuged again, then the sperm pellets were snap-frozen, and stored at
2808C for RNA or protein extraction procedures described below.
Effects of IL-6 and sIL-6R on sperm motility
All 20 sperm samples were diluted with serum-free HTF medium
with various concentrations of IL-6 (0 – 10 ng/ml, recombinant
human IL-6; Genzyme, USA) with or without sIL-6R (50 ng/ml;
recombinant human IL-6 soluble receptor; R&D Systems, USA).
The final concentration of sperm was adjusted to 5 £ 106/ml,
and 0.5 ml aliquots placed in 6 ml culture tubes (Falcon; Becton
Dickinson UK Ltd). A monoclonal antibody against IL-6R (5 mg/ml;
monoclonal anti-human IL-6 receptor antibody; Genzyme/Techne,
USA) was used to neutralize the specific effects of IL-6 and sIL-6R.
Each sample was incubated at 378C under a humid atmosphere of
5% CO2 in air. A part of the aliquots was evaluated by computer-
assisted sperm motion analysis (CASA; version 10 HTM-IVOS,
Hamilton Thorne Research, USA) system at 24 h. At the same time,
the sperm motility on the control samples was also assessed.
RT– PCR
Total RNA was extracted from the sperm by the guanidium thiocya-
nate method according to the manufacturer’s instructions (Isogen;
Nippon Gene Co. Ltd, Japan). Reverse transcription of RNA from
sperm into cDNA and PCR amplification were performed using the
Gene Amp RNA PCR Core Kit (Perkin Elmer, USA). The reverse
transcription of RNA to cDNA was performed with 2.5 IU/ml of
MuLV reverse transcriptase as follows: 10 min at 308C, 20 min at
47C, 5 min at 998C and 5 min at 48C. PCR amplification was carried
out in 25 mmol/l of a MgCl2 solution (2 mmol/l), 10 £ PCR buffer
II (1 £ ), DI water (65.5 ml) containing 0.1% DEPE, and Ampli Taq
DNA polymerase (2.5 U/100 ml). Samples were amplified for
32 cycles of denaturation (30 s at 948C), annealing (30 s at 608C),
1822
synthesis (1.5 min at 728C), and primer extension of 5 min at 758C
after each cycle.
For PCR analysis, previously described specific primers
for human IL-6R and human gp130 were used: IL-6R (sense):
50 -CATTGCCATTGTTCTGAGGTTC-30 and IL-6R (anti-sense):
50 -AGTAGTCTGTATTGCTGATGTC-30 ; gp130 (sense): 50 -ACAG-
ATGAAGGTGGGAAGGAT-30 and gp130 (anti-sense): 50 -AGATG-
ACATGCATGAAGACCC-30 (Yamazaki et al., 1996; Yoshioka
et al., 1999). The distances between primers, including the primers,
were 251 and 423 bp respectively. PCR products were resolved on
2% agarose gel with a small molecular weight DNA marker ( £ 174
digested with Hae III).
The PCR products were transferred to a nylon membrane (Sarton,
Germany) using a vacuum blotter with 0.4 mol/l NaOH and 1 mol/l
NaCl. The DNA on the membrane was fixed with a UV cross-linker.
The membranes were hybridized with internal probes of IL-6R
(50 -ACAAGCATGCATCCG-30 ) or gp130 (50 -CCAGATCCTTC-
AAAG-30 ). The biotin-labelled product on the membrane was
detected with a Smilight kit (Sumitomo Metal Industry Co., Japan).
The membrane was treated with streptavidin – alkaline phosphatase,
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followed by chemiluminescence detection. The membrane was
exposed to X-ray film for 15 min at room temperature (Iwabe et al.,
1998).
Western blot analysis
IL-6, IL-6R and gp130 protein expressions in sperm were examined
by means of western blot analysis as previously described in detail
(Yoshida et al., 2002). Briefly, the frozen sperm pellets were lysed
with lysis buffer containing 50 mmol/l Tris – HCl (pH 7.6),
150 mmol/l NaCl, 0.1% SDS, 1 mmol/l dithiothreitol, and 1 £ Com-
plete Protease Inhibitor Cocktail (Boehringer Mannheim, Germany).
The lysate was centrifuged and supernatant was prepared. Protein
concentration of the supernatant was measured by Bradford assay.
Eighty micrograms of protein samples were resolved by electrophor-
esis on a 4 – 20% gradient polyacrylamide gel. These separated pro-
tein samples were then electroblotted onto a nitrocellulose
membrane before blocking in 2.5% skimmed milk for 60 min. The
membrane was then probed with anti-IL-6R (1:1000) or anti-gp130
(1:1000) antibodies, (both from Genzyme/Techne) at 48C for 12 h
followed by a peroxidase-conjugated secondary antibody (1:1000).
Protein bands were visualized using an enhanced chemilumines-
cence system (Amersham, UK). The protein band size was deter-
mined using a Full Range Rainbow (Amersham) molecular weight
marker. In addition, the total protein from endometrial stromal cells,
which express both IL-6R and gp130 as previously described
(Yoshioka et al., 1999), was used as a positive control.
Statistical analysis
Results were analysed using two-way analysis of variance, followed
by Student’s paired sample t-test in order to compare the values
obtained with various treatments versus the values of control. The
data were expressed as means ^ SEM. P , 0.05 was accepted as
statistically significant.
Results
Effects of IL-6 and/or sIL-6R on sperm motility
To evaluate the effects of IL-6 and sIL-6R on sperm motility,
we examined Percoll-purified sperm fractions adjusted to
5 £ 106/ml (motility: 91.6 ^ 3.7%; and percentage rapid
cells: 66.8 ^ 6.9%, n ¼ 20). Neither IL-6 (10 ng/ml) alone
nor sIL-6R (50 ng/ml) added alone to the culture medium

Figure 1. Effect of IL-6, sIL-6R, and an anti-IL-6R antibody on
sperm motility and percentage of rapidly moving sperm. Sperm
(n ¼ 20) were cultured with IL-6 or sIL-6R, or with a combination
of both for 24 h. Sperm motility was evaluated using a computer-
assisted semen analysis (CASA) system. (A) Adding 10 ng/ml IL-6,
50 ng/ml sIL-6R, or 5 mg/ml of antibody against IL-6R (IL-6R Ab)
alone did not affect sperm motions (n ¼ 8). (B) The addition of IL-
6 (0.1 to 10 ng/ml) and sIL-6R (50 ng/ml) together adversely
affected sperm motility and percentages of rapidly motile cells in a
dose-dependent manner (n ¼ 12). Adding the anti-IL-6R antibody
abolished these adverse effects. *P , 0.05, **P , 0.001 versus
control.
had an effect on sperm motion. An antibody against IL-6R
also exerted no effect on sperm motility (Figure 1A). In con-
trast, as shown in Figure 1B, the addition of 0.1 –10 ng/ml
IL-6 and 50 ng/ml sIL-6R together adversely affected sperm
motility at 24 h culture. This combination of IL-6 and sIL-6R
also caused a profound decrease in the percentages of rapidly
moving sperm. The effect of IL-6 was dose dependent. Add-
ing the anti-IL-6R antibody abolished these adverse effects.
Agglutination of sperm was not observed following their
exposure to IL-6 plus sIL-6R.
Expression of IL-6, IL-6R, and gp130 in sperm
Because the addition of IL-6 or sIL-6R alone had no effect,
but, when combined, significantly reduced sperm motility,
we examined the gene expression of IL-6, IL-6R and gp130
in sperm by RT – PCR. A representative result of RT – PCR is
IL-6 and sperm motility
Figure 2. (A) RT– PCR detection of IL-6, IL-6R, and gp130 gene
expression in sperm. M: Size marker. Lane 1: GAPDH. Lane 2: IL-
6. Lane 3: IL-6R. Lane 4: gp130. (B) Specificities of PCR products
of gp130 were confirmed by hybridization of oligonucleotide probe.
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(C) Protein production of gp130 in sperm was detected by western
blot analysis. The total protein from endometrial stromal cells
served as a positive control. Lane 1: endometrial stromal cells. Lane
2: sperm.
shown in Figure 2A. Only a product of the expected size
(423 bp) of gp130 was detected. The identity of the PCR-
amplified cDNA fragment of gp130 was verified using
southern hybridization with the labelled internal probe
(Figure 2B). Furthermore, protein production of gp130 in
sperm was detected by western blot analysis (Figure 2C).
However, the IL-6 and IL-6R protein expressions were not
observed (data not shown).
Discussion
The present study demonstrates that the addition of combined
IL-6 and sIL-6R significantly reduced the percentage of
motile and rapidly moving sperm. On the other hand, the
addition of IL-6 or sIL-6R alone in the culture media failed
to adversely affect sperm motility. Sperm expressed only
gp130, and did not show the IL-6 and IL-6R expression,
suggesting that the combination of IL-6 and sIL-6R may
associate with gp130 and exert its effect on sperm.
For most cytokines (such as IL-2 and TNFa), soluble
receptors have antagonistic activity, precluding their ligand
binding to specific membrane receptors. In contrast, sIL-6R
is unique in exerting an agonistic action with its ligand,
resulting in subsequent signal transduction. Because gp130 is
expressed ubiquitously on various cells, IL-6 can exert its
biological effect in the presence of sIL-6R, even though
effector cells lack IL-6R on their plasma membrane, as
demonstrated by negative control of thyroid function in cul-
tured human thyroid follicles (Yamazaki et al., 1996). The
IL-6/sIL-6R complex associates with the constitutively
expressed gp130 on IL-6R-negative cells to induce homodi-
merization of gp130, leading to a cellular response. Thus, our
findings suggest an underlying mechanism in which IL-6
increases in the PF of patients with endometriosis, combines
with sIL-6R that also exists in PF, and the combined
1823
S.Yoshida et al.
IL-6/sIL-6R associate with the gp130 expressed on IL-6R-
negative sperm. Consequently, combined IL-6/sIL-6R may
exert adverse effects on sperm motility.
Furthermore, our data corroborate previous reports demon-
strating that IL-6 levels in seminal plasma or cervical mucus
were significantly higher in infertile patients compared with
the fertile control patients, and that its levels in semi-
nal plasma negatively correlated with sperm motility
(Gruschwitz et al., 1996; Naz and Butler, 1996). Since the
seminal plasma contains sIL-6R (Dousset et al., 1997),
increased IL-6 in seminal plasma and cervical mucus associ-
ate with sIL-6R, and then the binding of IL-6/sIL-6R com-
plex to gp130 presented in sperm may result in the
impairment of sperm function.
For the association of sperm motility and seminal plasma
IL-6 concentrations, some authors reported different results,
suggesting that IL-6 levels in seminal plasma did not corre-
late with sperm motility (Dousset et al., 1997; Kocak et al.,
2002; Friebe et al., 2003). The reasons for the controversy
are not evident but may reflect the effects of other factors
contained in seminal plasma. Seminal plasma contains var-
ious factors that stimulate or maintain sperm motility, such
as carnitine (Gurbuz et al., 2003), as well as inhibitory fac-
tors. The balance of those factors could regulate sperm
motions in the seminal plasma. We speculate that the effects
of the factors other than IL-6 may be responsible for the
controversy.
Seminal plasma reportedly contains similar levels of IL-6
with PF (Eggert-Kruse et al., 2001). In contrast, concen-
trations of IL-6 were much higher in cervical mucus of infer-
tile women than in PF of patients with endometriosis (Naz
and Butler, 1996). Indeed it seems that the sperm contact
with cervical mucus may be the most significant in vivo. The
exposure to elevated IL-6 levels in PF of patients with endo-
metriosis and in the Fallopian tubes subsequent to contact
with cervical mucus may further impair the sperm motility.
Additionally it is speculated that the seminal plasma, which
contains the protective factors of sperm motility, has been
removed from the fallopian tubes, and then IL-6 contained in
PF diffusing down the fallopian tubes may affect sperm
motions at lower concentrations than cervical mucus.
In the present study, we observed an inhibition of sperm
motility at a concentration of , 0.1 ng/ml of IL-6. The
median IL-6 concentration in PF of patients with endometrio-
sis (measured in our previous study) was lower than levels
used in the present study (Harada et al., 1997a). Thus, this
study has only a limited ability to determine the cause of
endometriosis-associated infertility for all patients. However,
in general, half of endometriosis patients are reported to be
infertile. The inhibition of sperm motility by IL-6 may be
involved in the infertility of at least some patients with endo-
metriosis who have highly elevated levels of IL-6 in PF.
PF diffusing into the tubal and endometrial environment
may affect sperm and their interaction with the oocyte and
embryo development. Many authors have demonstrated that
the PF of patients with endometriosis has detrimental
effects on several steps in the reproductive process. On the
other hand, various substances, including interleukins, were
1824
reported to increase in the PF of infertile women with endo-
metriosis (Ramey et al., 1993; Harada et al., 2001). Recently,
some of these substances are believed to be a cause of endo-
metriosis-associated infertility. In fact, substances such as
IL-1 (Sueldo et al., 1990), tumour necrosis factor-a (Eiser-
mann et al., 1989), endometrial placental protein 14 (Oehnin-
ger et al., 1995), antibodies to transferrin and a2-HS
glycoprotein (Pillai et al., 1998) were reported to adver-
sely affect sperm motility and function and/or embryo
development.
We previously reported that IL-6 was increased in the PF
of patients with endometriosis and correlated with the extent
of active endometriotic lesions (Harada et al., 1997a). We
also demonstrated that IL-6 was produced by endometriotic
stromal cells at levels similar to those of peritoneal macro-
phages from patients with endometriosis (Tsudo et al., 2000).
One study demonstrated that the addition of IL-6 to culture
medium reduced the blastocyst formation rate of mouse
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embryos, suggesting that increased IL-6 in the PF of endome-
triosis patients may contribute to infertility (Harada et al.,
1997b).
Thus, the concept that IL-6 may contribute to endometrio-
sis-associated infertility has been conjectural so far. The pre-
sent study supports earlier studies that demonstrated the
inhibitory effects of PF of patients with endometriosis on
sperm motility (Aeby et al., 1996) and functions (Coddington
et al., 1992; Arumugam, 1994), and it supports the contri-
bution of IL-6 to endometriosis-associated infertility by
adversely affecting sperm motility. From our series of studies
on IL-6, we conclude that IL-6 may contribute to the patho-
genesis of endometriosis-associated infertility.
Acknowledgements
We wish to thank Ms Taeko Kadowaki and Ms Miya Koike for
their technical assistance.
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Submitted on January 5, 2004; accepted on April 22, 2004
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