ORIGINAL ARTICLE
Incidence of Sperm-immobilizing Antibodies in Infertile Women
with Past Chlamydia trachomatis Infection
Yuki Hirano, Hiroaki Shibahara, Junko Koriyama, Makoto Tokunaga, Kazuhiko Shimada, Mitsuaki Suzuki
Department Obstetrics and Gynecology, School of Medicine, Jichi Medical University and Center for Reproductive Medicine, Jichi Medical University
Hospital, Tochigi, Japan
Keywords
Antisperm antibody, Chlamydia trachomatis,
infertility, sperm-immobilizing antibody
Correspondence
Hiroaki Shibahara, Department Obstetrics and
Gynecology, School of Medicine, Jichi Medical
University and Center for Reproductive
Medicine, Jichi Medical University Hospital,
3311-1 Yakushiji, Shimotsuke, Tochigi
329-0498, Japan.
E-mail: sibahara@jichi.ac.jp
Submission March 2, 2010;
accepted April 28, 2010.
Citation
Hirano Y, Shibahara H, Koriyama J, Tokunaga
M, Shimada K, Suzuki M. Incidence of sperm-
immobilizing antibodies in infertile women
with past Chlamydia trachomatis infection.
Am J Reprod Immunol 2011; 65: 127–132
doi:10.1111/j.1600-0897.2010.00883.x
Introduction
Antisperm antibodies are produced in both women1
and men.2 Several assay methods were developed to
detect antisperm antibodies. Among them, the sperm
immobilization test (SIT), which detects sperm-
immobilizing antibodies, has been shown to be the
American Journal of Reproductive Immunology 65 (2011) 127–132
ª 2010 John Wiley & Sons A/S
Problem
Among the risk factors for antisperm antibody production, inflammatory
diseases of the genital tract are believed to play an important role. Chla-
mydia trachomatis infection is one of the most common sexually trans-
mitted diseases. There are some reports suggesting that human sperm
have antigens that cross-react immunologically with certain microbial
antigens, such as C. trachomatis. However, this is still controversial. We
performed a retrospective study to investigate the correlation between
anti-chlamydial antibodies and sperm-immobilizing antibodies in infer-
tile women.
Method of study
Between January 2007 and March 2009, the presence of sperm-immobi-
lizing antibodies was examined by the sperm immobilization test using
sera from 273 infertile women. Anti-chlamydial antibodies (IgG and
IgA) were examined to prove past C. trachomatis infection by ELISA
using the same sera from infertile women.
Results
The overall incidence of sperm-immobilizing antibodies was 2.9%
(8 ⁄ 273) in infertile women. The incidences of sperm-immobilizing anti-
bodies were 6.4% (5 ⁄ 78) in cases with past C. trachomatis infection and
1.5% (3 ⁄ 195) in cases without past C. trachomatis infection. There was a
significant difference between the two groups (P = 0.031).
Conclusion
A significantly higher incidence of sperm-immobilizing antibodies was
noted in infertile women with past C. trachomatis infection compared
with that of those without past C. trachomatis infection. This is the first
demonstration that C. trachomatis infection could play a role in the pro-
duction of sperm-immobilizing antibodies in infertile women.
most reliable assay for detecting antisperm antibodies
closely related to female infertility.3,4 This observa-
tion was confirmed by other studies.5–8
The possible roles of sperm-immobilizing anti-
bodies in infertility are well known.1 However, the
factors that affect the production of sperm-immo-
bilizing antibodies in infertile women are not fully
127
 HIRANO ET AL.
understood. Moreover, the reason why most women
do not develop an immune response upon exposure
to sperm is not yet clear. Women do not generally
produce antibodies against sperm; however, some
infertile women have been found to possess anti-
sperm antibodies, which may contribute to their
infertility. Therefore, the following questions arise:
who produces sperm-immobilizing antibodies, what
makes women produce these antibodies, and what
are the corresponding antigens?
Among the risk factors for antisperm antibody pro-
duction, inflammatory diseases of the genital tract
are believed to play an important role as a conse-
quence of local inflammation, which was first sug-
gested in infertile men by Witkin and Toth.9 Later, it
was suggested that there is a very significant correla-
tion between antisperm antibodies and anti-
chlamydial antibodies in female oral contraceptive
users.10 It was also found that genital tract infec-
tions, including cervicitis caused by Chlamydia tracho-
matis, might lead to immunopotentiation of
antisperm antibodies that could affect fertility.11
Recently, it was shown that infertile couples with
genital C. trachomatis infection had higher incidences
of antisperm antibodies.12 These reports suggest that
C. trachomatis infection might play a role in the
induction of antisperm antibodies in women; how-
ever, the association between C. trachomatis infection
and the production of sperm-immobilizing antibod-
ies, especially those assessed by quantitative-SIT,13,14
has not yet been clearly understood.
We performed a retrospective study to investigate
the correlation between anti-C. trachomatis antibodies
and sperm-immobilizing antibodies in infertile
women.
Materials and methods
SIT and Quantitative SIT
Serum samples were collected with informed con-
sent from 273 infertile women between January
2007 and March 2009. All sera were heat treated at
56C for 30 min to inactivate them and were kept
frozen at )20C until use.
SIT was performed as previously described by Isoj-
ima et al.3,4 In brief, 10 lL of inactivated patient’s
serum, 2 lL of guinea pig serum (C’H50 = 200), and
1 lL of human sperm suspension (40 · 106 ⁄ mL)
were mixed on a Terasaki microplate and incubated
at 32C for 3 hr. As a control, the same amount of
128
heat-inactivated guinea pig serum was used instead
of the active complement. The sperm immobilization
value (SIV) was calculated by dividing the sperm
motility of the control serum by that of the test
serum. An SIV of two or more was considered posi-
tive for the SI antibody. Afterward, all sera with SI
antibodies were tested to determine the antibody
titers (SI50) by quantitative SIT as described by Isoj-
ima et al.13 and Koyama et al.14. The quantitative
SIT was used to reconfirm the SIT results by testing
for false-positive cases in the results of SIT. The SI50
titers are also clinically valuable for determining the
strategy for the treatment of infertile women.1
Anti-Chlamydia trachomatis Antibody Testing
Anti-C. trachomatis antibodies were examined to
prove past C. trachomatis infection by enzyme-linked
immunosorbent assay (ELISA; Peptide Chlamydia
IgA and IgG; Labsystems Oy, Helsinki, Finland) as
we described previously.15–17 Antibody titers >0.90
were considered as positive.
Statistical Analysis
Statistical analysis of the data was performed by Stu-
dent’s t-test using spss ver 17.0 (SPSS Inc., Chicago,
IL, USA) for Macintosh, and P < 0.05 was considered
significant.
Results
Overall Incidences of Sperm-immobilizing
Antibodies and Anti-Chlamydia trachomatis
Antibodies
The presence of sperm-immobilizing antibodies as
well as anti-C. trachomatis antibodies was examined
using sera from 273 infertile women. The overall
incidences of sperm-immobilizing antibodies and
anti-C. trachomatis antibodies were 2.9% (8 ⁄ 273) and
28.6% (78 ⁄ 273), respectively.
Comparison of the Incidences of Sperm-
immobilizing Antibodies Between Infertile Women
with and Without Past Chlamydia trachomatis
Infection
As shown in Fig. 1, the incidences of sperm-immobi-
lizing antibodies were 6.4% (5 ⁄ 78) in cases with past
Ct infection and 1.5% (3 ⁄ 195) in cases without past
American Journal of Reproductive Immunology 65 (2011) 127–132
ª 2010 John Wiley & Sons A/S
 INCIDENCE OF SPERM-IMMOBILIZING ANTIBODIES IN INFERTILE WOMEN
Fig. 1 Comparison of the incidences of sperm-immobilizing antibodies
between infertile women with and without past Chlamydia trachoma-
tis infection. The overall incidence of sperm-immobilizing antibodies
was 2.9% (8 ⁄ 273) in infertile women. The incidences of sperm-immobi-
lizing antibodies were 6.4% (5 ⁄ 78) in cases with past Ct infection and
1.5% (3 ⁄ 195) in cases without past Ct infection. There was a significant
difference between the two groups (P = 0.031).
Ct infection. There was a significant difference
between the two groups (P = 0.031).
Relationship Between Sero-positivity of Sperm-
immobilizing Antibodies and Anti-Chlamydia
trachomatis Antibody Titers in 78 Infertile Women
with Past Chlamydia trachomatis Infection
Anti-C. trachomatis antibody titers (IgA, IgG) were
compared between five women with sperm-immobi-
lizing antibodies and 73 women without the anti-
bodies (Table I). For anti-C. trachomatis antibody
(IgA), the titers were 1.48 ± 0.5 and 2.64 ± 0.4 in
Table I Relationship Between Sero-positive of Sperm
Immobilizing (SI) Antibodies and Anti-Chlamydia trachomatis
Antibody Titers in 78 Infertile Women with Past C. trachomatis
Infection
Anti-C. trachomatis
antibody titers
No.
SI-antibodies of patients IgA IgG
Positive 5 1.48 ± 0.5* 2.72 ± 1.0*
Negative 73 2.64 ± 0.4* 3.72 ± 0.4*
*P > 0.05.
American Journal of Reproductive Immunology 65 (2011) 127–132
ª 2010 John Wiley & Sons A/S
women with and without sperm-immobilizing
antibodies, respectively. There was no significant
difference between the two groups (P > 0.05). For
anti-C. trachomatis antibody (IgG), the titers were
2.72 ± 1.0 and 3.72 ± 0.4 in women with and with-
out sperm-immobilizing antibodies, respectively.
There was also no significant difference between the
two groups (P > 0.05).
Relationship Between Quantitative Value of
Sperm-immobilizing Antibodies (SI50 titers) and
Past Chlamydia trachomatis Infection in Eight
Infertile Women with Sperm-immobilizing
Antibodies in Their Sera
SI50 titers were compared between five women with
past C. trachomatis infection and three women with-
out the infection (Table II). SI50 titers in women
with any anti-C. trachomatis antibodies were
4.72 ± 2.9 and those in women without anti-C. tra-
chomatis antibodies were 5.30 ± 2.5. There was no
significant difference between the two groups
(P > 0.05).
Discussion
It is very important to understand why and how
some women produce antisperm antibodies, which
react with human sperm, and how the immune
reaction interferes with reproductive success,
because the development of new contraceptives is
necessary as current forms of birth control are
unavailable in some developing countries owing to
sociological, financial, or educational limitations.
The sperm-immobilizing antibodies, detected by
complement dependent SIT in the sera of infertile
women, were found to have a good correlation with
female infertility.3,4 It has been clearly shown that
Table II Relationship Between Quantitative Value of Sperm
Immobilizing Antibodies (SI50 titers) and Past Chlamydia
trachomatis Infection in Eight Infertile Women with Sperm
Immobilizing Antibodies in Their Sera
Anti-C. trachomatis No.
antibodies of patients SI50 titers
Positive 5 4.72 ± 2.9*
Negative 3 5.30 ± 2.5*
*P > 0.05.
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 HIRANO ET AL.
infertile women with sperm-immobilizing antibodies
are less likely to conceive because of inhibition of
sperm migration in the genital tract,18,19 as well as
blocking effects on the fertilization process.20–23
However, the factors that affect the production of
sperm-immobilizing antibody in some women are
not fully understood. Moreover, the reason why
most women do not develop an immune response
upon exposure to sperm is not yet clear. Our group
has already determined that the production of
sperm-immobilizing antibody in infertile women is
significantly related to HLA-DRB1*0901 and HLA-
DQB1*0303, indicating that only a few women can
immunologically respond to sperm antigens.24
Another concern focuses on the factor that makes
women produce sperm-immobilizing antibodies.
Using severe combined immunodeficient mice that
were heterotransplanted with human peripheral
blood lymphocytes from infertile women with
sperm-immobilizing antibodies, we previously
reported that human sperm themselves could be
responsible involving the immune response for the
production of sperm-immobilizing antibodies.25 Fur-
thermore, we recently investigated whether the pro-
duction of sperm-immobilizing antibodies in infertile
women is associated with their husbands’ sperm
count.26 It was found that the production of sperm-
immobilizing antibodies in women began after they
have been exposed to a large amount of sperm.
There are some reports suggesting that human
sperm have antigens that cross-react immunologi-
cally with certain microbial antigens, such as those
of C. trachomatis.9–12 However, this is still controver-
sial. Chlamydia trachomatis infection has recently
become the most common genital infectious disease
in the world.27 This pathogen causes urethritis and
epididymitis in men, and cervicitis and pelvic inflam-
matory disease in women. When asymptomatic cer-
vicitis develops into a persistent infection without
treatment, inflammation involving the Fallopian
tubes causes tubal stenosis, occlusion, and adhesion,
resulting in tubal factor infertility in approximately
10% of patients.28 Chlamydia trachomatis is also asso-
ciated with nidation disturbance in the prenidation
phase, spontaneous abortion in the late phase,29 and
in vitro fertilization-related embryonic development
disorders30,31 in addition to tubal damage. Even
when pregnancy is achieved, the presence of tubal
damage can result in ectopic pregnancy.32 Chlamydia
trachomatis infection in pregnant women induces
chorioamnionitis, leading to abortion or preterm
130
delivery in some cases.33 In other cases, infection of
the birth canal results in neonatal conjunctivi-
tis ⁄ pneumonia.34
Concerning C. trachomatis infection-related infertil-
ity, we previously reported that the incidences of
tubal passage disturbance and peritubal adhesion
were 32.3 and 59.7% in infertile women with a his-
tory of C. trachomatis infection, respectively.17
We performed a retrospective study to investigate
the correlation between anti-C. trachomatis antibodies
and sperm-immobilizing antibodies in infertile
women to determine whether C. trachomatis infection
induces the production of sperm-immobilizing anti-
bodies in women, because the association between
them has not yet been clearly understood.
As shown in Fig. 1, a significantly higher inci-
dence of sperm-immobilizing antibodies was noted
in infertile women with past Ct infection compared
with that of those without past Ct infection. This is
the first demonstration that Ct infection could play a
role in the production of sperm-immobilizing anti-
bodies in infertile women. Dimitrova et al.12 previ-
ously described that the genital C. trachomatis
infection might play a role in the induction of anti-
sperm antibodies. They used sera from women and
men and analyzed the data together, which might
confuse understanding of the sex difference of sperm
immunity. Antisperm antibody testing was per-
formed using gelatin agglutination test, tray aggluti-
nation test, SIT, and ELISA. However, they did not
carry out quantitative SIT to confirm SI50 titers,
which might cause a higher incidence of sperm-
immobilizing antibodies of 7.27% (4 ⁄ 55) in the sera
from healthy blood donors of proven fertility. This
may also have caused the markedly higher rate of
positive results (24.6%) of SIT than of ELISA
(10.7%) in the sera from infertile couples with geni-
tal C. trachomatis infection. Therefore, this study is in
fact the first to clarify the positive relationship
between C. trachomatis infection and the production
of sperm-immobilizing antibodies in women.
In this study, there were no significant differences
in anti-C. trachomatis antibody titers (IgA, IgG)
between infertile women with and without sperm-
immobilizing antibodies (Table I). There was also no
significant difference in SI50 titers between infertile
women with and without past C. trachomatis infec-
tion (Table II). These data suggest that one of the
triggers for the production of sperm-immobilizing
antibodies in women might be C. trachomatis infec-
tion. However, the production of sperm-immobiliz-
American Journal of Reproductive Immunology 65 (2011) 127–132
ª 2010 John Wiley & Sons A/S
 INCIDENCE OF SPERM-IMMOBILIZING ANTIBODIES IN INFERTILE WOMEN

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