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Surgical Science, 2011, 2, 31-37
doi:10.4236/ss.2011.21008 Published Online January 2011 (http://www.SciRP.org/journal/ss)
Copyright © 2011 SciRes. SS
Local Cell Mediated Immune Reaction in Primary
Obstructive Male Infertility
Anil Kumar Sarda, Shweta Aggarwal Bhalla, Durgatosh Pandey, Shyama Jain*
Departments of Surgery & Pathology*, Maulana Aza d Medical College & associated
L. N. Hospital, New Delhi, INDIA.
Received August 9, 2010; revised October 15, 2010; accepted January 13, 2011
Increased intra-epididymal pressure due to obstruction causes a breach in the epididymal lining exposing the
highly antigenic sperms to the interstitium. When the exposure is small and intermittent, it is likely to induce
a local humoral response which may affect sperm maturation and also react with the epididymal epithelium
producing irreversible histological changes. The present study on 30 patients of primary obstructive infertil-
ity found direct and indirect evidence of the production of antisperm antibodies locally. ELISA for antisperm
antibodies was positive in the epididymal fluid in 16/30 (53%) of the patients. Indirect evidence of the role of
local antigen-antibody reaction in the epididymis is apparent in 22/30 (73%) patients who had a lymphocytic
infiltrate. The local presence of antisperm antibodies in the epididymal fluid correlated well with the pres-
ence of lymphocytic infiltration in the interstitium (p < 0.05). In our study 16/30 patients had a positive
ELISA for antisperm antibody and all these patients had interstitial lymphocytic infiltration. In addition eight
patients with a negative ELISA also showed interstitial lymphocytic infiltration. Thus 22/30 (73%) patients
had an evidence of a local immune reaction directly in the form of a positive ELISA for antisperm antibodies
in the epididymal fluid and / or indirect evidence in the form of lymphocytic infiltrate in the interstitium.
None of the five controls had either a positive ELISA or lymphocytic infiltrate, this difference was found to
be statistically significant (p < 0.005).
Keywords: Male Infertility, Autoimmunity, Testis, Epididymis, Histology
The reported prevalence of antisperm antibodies (ASA)
varies depending on the modality of the immunological
screening. Circulating ASA detected with indirect tests
ranges from 8.1% to 30.3% in unselected men with in-
fertile marriages; at low titres they were also reported in
2.4% to 10% of fertile men . When stricter criteria
were used, the prevalence of ASA in men with infertile
marriages was 4.7% to 7.5%. Obstruction leading to the
production of antisperm antibodies is evidenced by the
fact that 34% – 74 % men develop antisperm antibodies
post vasectomy . Though the association of epididy-
mal occlusion with antisperm antibodies has not been
widely studied, a recent report shows that antisperm an-
tibodies are present in the serum of men with epididymal
occlusion, whether of infective or congenital origin. The
incidence is higher when occlusion is more distal .
During the onset of spermatogenesis at puberty, new
developmental antigens make their appearance on the
sperm surface . Since immune tolerance for
self-antigens is expressed neonatally, these newly ap-
pearing sperm antigens may be immunogenic. It has been
theorized that sequestration of developing sperm by the
blood testis barrier prevents the generation of autoanti-
bodies to sperm . Normally the epididymal epith elium
is well adapted to prevent transwall migration of immu-
nologically active material. However, in case of obstruc-
tive azoospermia, leakage of spermatozoal degradation
fragments occurring proximal to th e site of obstruction in
the epididymis , or trauma to the testis or a breach of
the male reproductive tract allows the sperm antigens to
escape into the microcapillaries surrounding the repro-
ductive tract and then into the systemic circulation and
stimulate a humoral immune response. Genital tract in-
fections can also induce antisperm antibodies production
A. K. SARDA ET AL.
Copyright © 2011 SciRes. SS
due to a breach in blood testis barrier due to inflamma-
tion or by production of antibodies against the infective
agents like Chlamydia which cross react with spermato-
zoa . Approximately 34% to 74% prevalence of ASA
is reported in men who have undergone a vasectomy,
with their persistence in 38% to 60% following success-
ful vasovasostomy  which may have a negative impact
The deleterious effect of antisperm antibodies on fer-
tility has been well documented [8-11]. Also, the epidi-
dymis is considered to be the most likely source of anti-
body secretion. It is populated with lymphocytes and
macrophages . The secretion of antibodies by the
epididymis may be increased in cases of obstructive in-
fertility as suggested by the presence of inflammatory
cells including plasma cells, in the interstitium of these
Increased intra-epididymal pressure due to obstruction
has been reported to cause a breach in the epididymal
lining exposing the highly antigenic sperms to the inter-
stitium. When the exposure is large antisperm antibodies
are produced in the serum. However, small, intermittent
exposure to sperms is likely to induce a local humoral
response which may not manifest itself in the serum.
These antisperm antibodies secreted locally in the epidi-
dymis would act on the already disintegrating spermato-
zoa and further impair their function and maturation. It is
possible that the antigen-antibody complexes also react
with the epididymal epithelium producing irreversible
histological changes, which impair the function of the
epidididymis, in turn impairing sperm maturation. Thus,
the persistence of infertility in patients with obstruction
to the egress of sperms even after a patent anastomosis
could be due to the abnormal morphology or impaired
function of the spermatozoa caused by locally secreted
antisperm antibodies. Further, with progress in assisted
reproductive techniques, the role of ASA in male infer-
tility is becoming better defined.
We believe that repeated exposure to small amounts of
extravasated sperms in the epid idymal interstitium would
be sufficient to produce antisperm antibodies locally
which could then initiate a local cell mediated immune
reaction with its attendant effects on the epididymis and
sperms. The role of local immune reaction in obstru ctive
infertility has no t been widely studied . The present study
focuses on the role of local cell mediated immune re-
sponse in primary obstructive male in fertility.
2. Materials and Methods
Thirty patients of primary obstructive infertility with
azoospermia, presence of fructose in the semen and nor-
mal spermatogenesis were included. Details of material
and methods have been described in an earlier report .
Six patients with bilateral absence of vas deferens were
also included in our study because of our policy to con-
firm absence of vas by scrotal exploration and in these
patients to an artificial spermatocele in these patients to
give them a chance for utilizing one of the cheaper me-
thods of assisted reproduction, i.e. intra-uterine insemi-
nation of aspirated sperms.
The remaining patients were subjected to a single
layer side-to-side vasoepididymostomy if the epididymal
ducts were dilated, microscopic examination of the epidi-
dymal fluid revealed the presence of sperms and the dis-
tal vas was patent . The epididymis was incised and
tissue taken for histo logical examination. The epididymal
fluid was taken with a micro-pipette was diluted with
normal saline for determination of anti-sperm antibodies
in the supernatant fluid with the precipitated sperms be-
ing used to study sperm morphology and other studies.
The procedure was completed after taking a testicular
biopsy which included part of tunica albuginea.
Five controls were also studied. They were of proven
fertility. Testicular biopsy and epididymal wedge biopsy
were taken at the time of autopsy or from patients who
underwent orchiectomy for any reason except for tes-
ticular torsion or testicular malignanc y. Epididymal fluid
was also aspirated for cytology for sperm morphology
and also for analysis of antisperm antibodies.
Grade of spermatogenesis in the testicular tissue was
done according to the accepted criteria . The inter-
stitium was specially screened for presence of lympho-
cytes, plasma cells, histiocytes. Mature spermatids will
be identified as oval cells with dark densely stained chro-
matin. The number of mature spermatids will be totaled
in twenty tubules and the total be divided by twenty to
give an estimate of number of mature spermatids per
tubule for quantitation of mature spermatids per tubule
The histologic features were recorded on a pre-deter-
mined performa as described earlier .
ELISA technique was used to detect antisperm anti-
bodies by utilizing a commercially available kit. The kit
is an indirect noncompetitive enzyme immunoassay for
the semiquantitative and qualitative determination of
antibodies directed to spermatozoa surface antigen. The
wells of a microtiter plate are coated with spermatozoa
surface antigen. Sperm specific antibodies present in the
patient sample bind to the antigen. In a second step the
antigen-antibody co mplex reacts with an enzyme labeled
second antibody (Enzyme Conjugate) which leads to the
formation of an enzyme labeled antigen-antibody sand-
wich complex. The enzyme label converts added sub-
strate to form a colored solution. The rate of color for-
mation from the chromogen is a function of enzyme
A. K. SARDA ET AL.
Copyright © 2011 SciRes. SS
conjugate complexed with the bound antibody and thus
is proportional to the initial concentration of the respec-
tive antibodies in the patient sample. The detection limit
of the assay is 0.2 U/ml and permits the determination of
lgG, IgM and lgA antibodies directed against sperm sur-
The Chi square test was used for statistical analysis
with Yates modification wherever applicable.
The age of the patients in the study group ranged from
24-49 years (mean: 31.1 yrs.; median: 30 yrs.). Forty
three percent of the patients were > 30 years old at pres-
entation. None of the patients had any documented his-
tory of genital infections or any history suggestive of
prior urinary tract infections. None had suffered from
Three patients had significant levels of antisperm an-
tibody levels in the serum. However, only one of these
patients had positive ELISA for antisperm antibodies in
the epididymal fluid. On the other hand, 15 out of 27
patients with absence of antisperm bodies in blood had a
positive ELISA for presence of antibodies in the epidiy-
mal fluid. This finding was not found to be statistically
At operation the vas was absent in 5 pa tients (16.7%);
22/25 of the remaining patients had a patent vas deferens
(88%). Discharge on cutting the epididymal duct was
minimal (+) in 9 (30%), moderate (++) in 10 (33.3%),
profuse (+++) in 5 (16.7%) and absent in 6 (20%). In the
five patients with an absent vas epididymal discharge
was absent in 40%, minimal in 40% and moderate in
Epididymal distension correlated well with discharge
on cutting the epididymis. Thus, all the six patients with
no discharge on cutting the epididymis, did no t have dis-
tended epididymis although 7/13 patients without a dis-
tended epididymis still had discharge on cutting the epi-
didymis (1 + in 6 patients and 2 + in one patient). All
patients with distended epididymis had 1+ to 3 + dis-
charge on cutting the epididymis. However, the post op-
erative period is too short to assess the effect of these
findings on the outcome of the surgery.
ELISA for antisperm antibodies in the epididymal
fluid was found to be positive in 16/30 (53.3%) patients.
Negative results may have occurred in the remaining
patients either because of the low concentration or the
small amount of epididymal fluid available which was
further diluted by the saline in which it was collected.
Two of the patients with significant ELISA in the epidi-
dymal fluid had positive antisperm antibodies in the se-
The histological findings of the epididymis and their
correlation with ELISA results are represented in Table 1.
Dilatation of ducts, epithelial breach and sperm ex-
travasation can be considered a direct result of obstruc-
tion and was demonstrated in 63.3%, 43.3% and 26.7%
of the patients under study (Figures 1 & 2). Sperm in-
gestion by macrophages was reported in 33.3% of the
cases (Figure 3); in 8 cases epididymal discharge could
be obtained and of these 75% had significant ELISA.
Amongst the 22 cases with lymphocytic (majority
T-lymphocytes), macrophage and plasma cell infiltration
(Figures 4 & 5), 100% of the cases in whom epididymal
fluid could be obtained had significant ELISA. In 12/14
patients with loss of cilia there was associated ductal
dilatation; significantly 78.6% also had lymphocytic in-
filtration and in 60% the ELISA was significant.
None of the control group patients had any humoral or
cellular evidence of local immune reaction.
Thus, 22/30 patients had evidence of local immune
reaction in the form of interstitial inflammatory cell in-
filtrate in epididymal tissue and/or a positive ELISA for
antiserm antibodies in epididymal fluid while 0/5 con-
trols had evidence of local immune reaction. The differ-
ence between cases and controls was statistically signifi-
cant (p < 0.005).
Sperms are highly antigenic cells. During the onset of
spermatogenesis at puberty, new developmental antigens
make their appearance on the sperm surface . It has
been theorized that sequestration of developing sperm by
the blood-testis barrier prevents the generation of autoan-
Table 1. Effect of obstruction on the epididymal histology.
Histological findings Patients
(n = 30)
Dilatation of ducts 19 3 9/16
Loss of cilia 14 4 6/10
Presence of macrophages
in ducts 18 4 9/14
Sperm ingestion by ma-
crophages 10 2 6/8
Sperm extravasation 8 2 4/6
Epithelial breach 13 2 8/11
Lymphocytic and plasma
cell infiltration in inter-
stitium 22 6 16/16
in interstitium 4 0 3/4
A. K. SARDA ET AL.
Copyright © 2011 SciRes. SS
Figure 1. Photomicrograph of the epididymis showing di-
lated ducts with focal flattening of the epithelium (H&E
Figure 2. Photomicrograph of the epididymis showing epi-
thelial flattening and breach, and extravasation of sperms.
Figure 3. Photomicrograph of the contents of the dilated
epididymal ducts. Macrophages with phagocytosed sperms
and desquamated lining cells are seen. (H&E x400)
tibodies to sperm . The products of sperm break-
Figure 4. Photomicrograph of the epididymis showing
sperm extravasation with interstitial cell infiltration (H&E
Figure 5. Photomicrograph of the epididymis showing in-
terstitial collection of macrophages with a dilated epididy-
mal duct to the left. (H&E x400)
down on absorption would be an immense immunelogcal
stimulus leading to immune response. However, T-lym-
phocytes, representing 12.7% of the mucosal cell popu-
lation of the epididymal epithelium  act as the im-
munological suppressor barrier preventing such a re-
sponse from occurring . Sperms are also segregated in the
epididymis due to the presence of the tight junction
complexes in the lining epithelium of the epididymis
Which p r ev en t th e le ak ag e of sperm an tig ens and pr event
their contact with local and circulating immune effector
cells Any breach in the reproductive tract due to the v ar-
ious factors leads an antibod y response against th e sperm.
The two major classes of antisperm antibodies are IgG
and IgA. IgG is a systemic immunoglobulin that is se-
creted in a variety of tissues and fluid compartments. IgA,
on the other hand, is a secretory antibody whose produc-
tion may be locally mediated. Significant levels of IgM
do not reach the reproductive tract fluid in the male and
A. K. SARDA ET AL.
Copyright © 2011 SciRes. SS
it is unlikely that IgM an tibodies contribute to infertility.
We studied the antisperm antibodies (IgA, IgG and IgM)
in the serum of our patients and found that 3/30 patients
were positive for the same. de Kretser et al have reported
that antisperm antibodies occur more frequently in the
serum of men with obstructive infertility than th e normal
population . This finding was confirmed in our study
where 3/30 (10%) of our patients had positive serum
antisperm antibodies as compared to 2% in the normal
fertile men. Various authors have studied the effect of
obstruction on the epididymal histology and on the
sperms. Rajalakshmi et al found degenerative changes
due to pressure effects , while McConnel observed
fibrosis, sperm granuloma and interstitial macrophages
. Phadke observed epithelial breach, sperm extrava-
sation, macrophages and interstitial inflammatory cells
. Hargreave et al observed interstitial infiltration by
lymphocytes, macrophages and plasma cells with fibrosis
and perivasculitis . Phadke also observed ingestion
of sperms by macrophages . Rajalakshmi et al ob-
served a higher percentage of morphological abnormali-
ties and poor motility . The commonest abnormal
forms were sperms with pyriform head, the others being
round, tapering, long and large heads as well as abnor-
malities in midpiece and tail.
We believe that breach of epididymal epithelium
causes extravasation of sperms into the interstitium and
brings then into contact with the antibody producing
cells. If the exposure is not large enough to reach the
lymph nodes, antisperm antibodies will not result in the
serum. However, the exposure would be sufficient to
produce antisperm antibodies locally which could then
initiate a local cell mediated immune reaction with its
attendant effects on the epididymis and sperms.
Direct evidence for the production of antisperm anti-
bodies locally was obtained in the present study where
we found that ELISA for antisperm antibodies was posi-
tive in the epididymal fluid in 16/30 (53%) of our pa-
tients. This is an important observation establishing the
role of local antigen-antibody reaction in patients of pri-
mary obstructive infertility. If taken into conjunction
with the fact that only three of the patients under study
had raised antisperm antibodies in the serum, out of
which only two patients had a positive ELISA for antis-
perm antibodies in the epididymal fluid, this finding as-
sumes further significance in establishing the importance
of local production of antisperm antibodies in the epidi-
dymis. We were unable to access any reported literature
on this subject.
Indirect evidence of the role of local antigen-antibody
reaction in the epididymis is app arent in the observations
on epididymal biopsy. We found that 22/30 (73%) pa-
tients showed a lymphocytic and plasma cell infiltrate in
the interstitium which is an indirect evidence of the oc-
currence of an immune reaction. The local presence of
antisperm antibodies in the epididymal fluid correlated
well with the presence of lymphocytic and plasma cell
infiltration in the interstitium (p < 0.05). In our study
16/30 patients had a positive ELISA for antisperm anti-
body and all these patients had interstitial lymphocytic
and plasma cell infiltration. In addition eight patients
with negative ELISA also showed interstitial lympho-
cytic and plasma cell infiltration. Thus 22/30 (73%) pa-
tients had an evidence of a local immune reaction di-
rectly in the form of a positive ELISA for antisperm an-
tibodies in the epididymal fluid and/or indirect evidence
in the form of lymphocytic and plasma cell infiltrate in
the interstitium. None of the five controls had either a
positive ELISA or lymphocytic infiltrate, this difference
was found to be statistically signif icant (p < 0.005).
Thus, we found a definite evidence of the presence of
a local autoimmune reaction in patients of primary ob-
structive infertility included in our study. It is possible
that in six patients where lymphocyte and plasma cell
infiltration was present but ELISA for antisperm anti-
body was negative and those cases with macrophage
infiltration but negative ELISA, the local secretion of
antibodies may have been in low concentration and was
not detected by ELISA.
The local antibody response was not studied in previ-
ous reports of epididymal histology in obstructive infer-
tility [13,18]. Phadke reported epithelial breach with
sperm extravasation in patients of obstructive infertility
. He also found increased number of intraluminal and
interstitial macrophages and suggested that these cells
present the antigens to the local lymphatic sites where
antibodies against spermatic antigens are produced. He
suggested that it is likely that in cases of obstructive in-
fertility some antigenic components of spermatozoa are
also absorbed through intact epithelium and transferred
to basal capillaries which may be responsible for devel-
opment of autoantibodies against spermatozoa. Other
authors also in their studies of the effect of obstruction
on the epididymal histology have found an increased
interstitial cell inflammatory infiltrate in cases of ob-
structive azoospermia which is an evidence of the pres-
ence of a local immune reaction [13,18,19], Phadke re-
ported macrophage infiltration in the interstitium in 8/32
cases and perivascular accumulation of plasma cells in
two cases , while a lymphocytic infiltration and fi-
brosis was reported by McConnel . These are pre-
sumptive evidence of the presence of an antigen – anti-
body reaction although a direct evidence of the presence
local antibodies in these cases was not obtained. Since
the histologic findings of the epididymis in their reports
are similar to those in the presen t stud y, it is po ssible that
A. K. SARDA ET AL.
Copyright © 2011 SciRes. SS
they also resulted from a local autoimmune reaction.
However, lack of direct evidence of the presence of an-
tisperm antibodies in the epidid ymal fluid precludes from
coming to any positive conclusion regarding this aspect.
In our study we found intraluminal macrophages in 18/30
cases and 9/18 of these cases had a positive ELISA for
antisperm antibodies. Although this finding is statisti-
cally insignificant, the negative ELISA could be ex-
plained by the presence of antibodies in low titres in
Thus there is enough evidence to suggest that a cell
mediated immune response occurs in males with primary
obstructive infertility and produces changes in both the
luminal and extraluminal portio ns of the epididymis. The
local immune response is likely to impair fertility through
various likely mechanisms. In most epidemiologic stud-
ies there is little evidence that suggests a cause/effect
relationship between ASA and abnormality of the prin-
cipal semen parameters (sperm count, motility and mor-
phology) between infertile patients with and without
ASA . Sperm injury mediated by complement medi-
ated sperm cytotoxicity is potentially possible. Although
anticomplementary activity has been reported in human
semen, this mechanism has been documented in the fe-
male genital tract [1,21]. Antisperm antibodies and com-
plement deposition resulted in a dramatic loss of sperm
motility, as well as in activation and aggregation (roset-
ting) of polymorphonuclear leukocytes (PMN) to anti-
body- and complement-bound sperm . Several studies
suggest that ASA can interfere with sperm functions in-
volved in the fertilization process by b locking sperm-egg
interactions. This would affect natural fertilization and
even in assisted reproductive techniques it has to be
taken into account both as an interfering factor blocking
sperm-egg interactions and also as a complicating factor
due to ASA production after insemination .
The on-going stud y was conducted on behalf of and with
grants-in-aid from the Department of Family Welfare,
Ministry of Health and Family Welfare, Government of
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