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- Original Article -
Follicle-stimulating hormone autoantibody is involved in
idiopathic spermatogenic dysfunction
Bing Yao1, Jian Wang1*, Wei
Liang2*, Ying-Xia Cui1, Yi-Feng Ge1
1Institute of Clinical Laboratory Medicine, Nanjing Jinling Hospital, Nanjing University, Nanjing 210002, China
2Department of Chinese Traditional Medicine, No.454 Hospital, Nanjing 210002, China
Abstract
Aim: To detect the anti-follicle-stimulating hormone (FSH) antibody in idiopathic infertile patients and fertile subjects
in order to determine the role of this antibody in patients with spermatogenic dysfunction.
Methods: The anti-FSH antibody in serum was detected by an enzyme-linked immunosorbent assay (ELISA). The functional and structural
integrity of the sperm membrane was evaluated with hypo-osmotic swelling (HOS) test and the ultrastructure of the
spermatozoa was investigated by transmission electron microscopy (TEM).
Results: The extent of positive FSH antibody in the patients with oligozoospermia and/or asthenozoospermia was significantly higher than that in the fertile
subjects and infertile patients with normal sperm concentration and motility, but it was significantly lower than that in
the patients with azoospermia. The extent of anti-FSH antibody in the patients with azoospermia was significantly
greater than that in patients with oligospermia and/or asthenospermia, infertile people with normal sperm density and
motility and fertile people. The hypo-osmotic swelling test showed that the percentage of HOS-positive spermatozoa
(swollen) was 45.1% ± 3.5% in the FSH antibody-positive group and 59.1% ± 6.2% in the FSH antibody-negative
control group. The percentage of functional membrane damage to spermatozoa was significantly higher in the
anti-FSH antibody-positive group than in the control group. TEM showed that the outer acrosomal membrane was located
far from the nucleus, and detachment of the acrosome was found in the FSH autoantibody-positive
group. Conclusion: These data suggest that the presence of anti-FSH antibody is strongly correlated with the sperm quantity and quality
in idiopathic male infertility. Anti-FSH antibody may be an important factor causing spermatogenic dysfunction and
infertility. (Asian J Androl 2008 Nov; 10: 915_921)
Keywords: follicle-stimulating hormone; antibody; reproduction; spermatogenic dysfunction
Correspondence to: Dr Yao Bing, Institute of Clinical Laboratory Medicine,
Nanjing Jinling Hospital, Clinical School of Medical College, Nanjing
University, Nanjing 210002, China.
Tel: +86-25-8086-0174 Fax: +86-25-8481-5775
E-mail: yaobingliang@yahoo.com.cn
*These authors contributed equally to this work.
Received 2008-06-02 Accepted 2008-07-07
DOI: 10.1111/j.1745-7262.2008.00441.x
1 Introduction
Follicle-stimulating hormone (FSH) is one of the
pituitary hormones that controls fertility in both males and
females. For the male, FSH is essential for
spermatogenesis [1] and many studies have demonstrated the
biological role of FSH in rodent testes. For example, Singh
and Handelsman [2] administered testosterone (T) and
exogenous FSH to the gonadotrophin-releasing hormone
(GnRH)-deficient mouse and found that when treated with
testosterone they had normal spermatogenesis but the
testicular size and germ cell numbers were reduced. In
contrast, GnRH-deficient mouse treated with FSH had
quantitatively normal spermatogenesis as well as normal
size of the testis. The size of testis was increased by
43% after the mouse was treated with exogenous FSH
[2]. Several other studies on gene mutations have
confirmed this observation. For example, males with
FSHβ gene mutations show the symptoms of azoospermia and
their periods of puberty can be normal or absent.
Furthermore, the testes of FSH null mutants were
approximately half the normal size and the number of
epididymal spermatozoa was reduced by 75% at the age of 6_7
weeks [3]. Further studies showed that injection of
FSH-specific polyclonal antibody into pregnant hamsters on
the 12th, 13th or 14th day of gestation or into newborn
hamsters significantly reduced the number of primordial
follicles. These results suggested that anti-FSH antibody
inhibits the critical role of FSH on the formation of
primordial follicle formation during fetal ovarian
development [4]. Even though the role of anti-FSH antibody has
been experimentally demonstrated in animals, it is still
unclear whether FSH autoantibody is present and involved
in the idiopathic infertilities with spermatogenic
dysfunc-tion. Therefore, in the present study, we investigated
the presence of FSH autoantibody in idiopathic
infertilities with spermatogenic dysfunction and in fertile people.
In addition, we evaluated the functional and structural
integrity of sperm membrane and sperm ultrastructure
in FSH antibody-negative and -positive groups.
2 Materials and methods
2.1 Reagents
FSH and luteinizing hormone (LH) from human pituitaries were purchased from Sigma (St. Louis, MO,
USA). FSH, LH, T and prolactin (PRL)
radioimmunoassay kits were purchased from Beijing Fu Rui
Biotechnology (Beijing, China). Polystyrene microplates were
purchased from Combiplate 8, Biohit (Helsinki, Finland).
Anti-FSH serum was purchased from Calbiochem (La Jolla, CA, USA). The anti-sperm antibody detection kit
was produced by Rui Di Bioproduction (Nanjing, China).
2.2 Patients
The idiopathic infertility group included 150 patients
who visited the Andrology Department at Nanjing
Jing-ling Hospital between 2004 and 2007 (age 27.9 ± 4.6
years). A team consisting of urologists and andrologists
performed detailed clinical investigations of all the
patients and recorded the complete case history of each
individual. All the infertile men were also subjected to
karyotyping and endocrinological assays. The testicular
volume of each patient was determined with
punched-out elliptical rings (Takahara orchidometer). The
volume of each testis was from 18.5 mL to 33.4 mL. Patients
with non-obstructive azoospermia were confirmed with
the observation of all phases of spermatogenic cells in
testicular biopsy, thus excluding the obstruction in
seminiferous tubule. The levels of serum FSH (1.42_15.2
U/L), LH (l.2_7.8 U/L), T (0.52_38.17 nmol/L) and PRL
(1.3_3.5 mIU/L) in all the samples of this study were normal.
Y chromosome microdeletions in the AZF (azoospermic
factor) regions, as described by Imken [5], were not
found. Anti-sperm antibody was not detected in all the
patients. A control group included 50 men (age 28.7 ±
3.9 years) whose partners had borne their children within 1
year of marriage and had normal sperm concentration
and motility.
2.3 Semen samples and analysis
Human ejaculated spermatozoa were obtained by masturbation after 3 days of sexual abstinence. Semen
samples were liquefied at room temperature within
20_30 min of delivery. Semen samples were analyzed by
placing a volume of 5 μL semen into a disposable semen
analysis chamber (Cell-Vu; Fertility Technologies, Natick,
MA, USA). Semen analysis was performed according to World Health Organization (WHO) criteria (1999) [6]
All semen samples were analyzed by the same technician.
Motility was analyzed for at least 200 sperm of each
sample. The motility of each spermatozoon was graded
as `a', `b', `c' and `d' according to the following criteria:
(a) rapid progressive motility, (¡Ý 20 μm/s); (b) slow or
sluggish motility; (c) non-progressive motility (< 5
μm/s); and (d) non-motile. Sperm motility was calculated as
the number of spermatozoa with grade a + b motility
divided by the total number of sperm (× 100). A repeat
sperm motility assessment was performed on a separate
aliquot of 5 μL from the same semen sample. If the two
measures of sperm motility from the same specimen varied beyond the 95% confidence interval (CI) [6], two
new slides were prepared and sperm motility was reassessed. When the two measures were within the
95% CI, the two measures were averaged and reported
in this study. Patients were diagnosed with infertility
and characterized as oligozoospermia, asthenozoospermia
and azoospermia according to Layman [7]. Among 150
patients, 98 cases suffered from oligozoospermia (the
concentration of spermatozoa was less than 20 million/mL)
and/or asthenozoospermia (the motility of the
spermatozoa was less than 50%), 22 patients suffered from
azoospermia (the absence of spermatozoa) and 30 patients suffered from infertility with normal sperm
concentration and motility.
2.4 Enzyme-linked immunosorbent assay (ELISA)
The serum samples were acupunctured from the
brachial vein and centrifuged for 30 min and immediately
frozen at _20ºC. Serum anti-FSH antibody level was
determined by an antibody-captured ELISA using purified
human pituitary FSH. A preliminary experiment was
performed to determine the optimal conditions for FSH
detection. After chessboard titration, we found that
positive control serum diluted 1:100 and polystyrene
microplates coated with 0.15 pmol purified FSH were
the optimal conditions. After blocking, 100 μL of the
diluted serum samples were added into the microplates,
coated with FSH and incubated at 37ºC for 30 min.
After washing with phosphate buffered saline-Tween-20
(PBST) (pH 7.2), 100 μL of horseradish peroxidase (HRP)-conjugated goat-anti-human immunoglobulin G
(IgG; 1 : 2 000) was added and incubated at 37ºC for 30
min. The plates were then washed thoroughly with PBST and
the absorbance (A) at 450 nm was measured after 100
μL of tetramethyl benzidine-hydrogen peroxide (TMB/
H2O2) substrate was added and incubated at room temperature
for about 10 min. A known negative control serum sample
and a blank (0.01 mol/L PBS, pH 7.4) were included in
each assay and the positive sample was judged as the
value of (A of tested sample _ A of blank)/(A of negative
control _ A of blank) ¡Ý 2.1 [8].
Anti-FSH serum was also included in each assay. The
performance of all antibodies was determined under an
internal quality assessment scheme [9].
The positive samples were verified by another ELISA
assay using 100 μL of synthetic peptide (0.5 ng/mL)
synthesized by HD Biosciences (Shanghai, China) as the antigen.
This peptide corresponds to the sequence of the 33-53 amino
acid region of human FSH beta-chain and th e sequence of
the synthesized peptide is EECRFCISINTTWCAGYCYTR.
Samples were accepted as positive when both ELISA assays indicated positive detection of anti-FSH.
2.5 Hypo-osmotic swelling (HOS) test
The HOS test was performed according to the method described by Jeyendran
et al. [10]. Briefly, 0.1 mL sperm suspension was mixed with 1 mL hypo-osmotic
solution (equal parts of 150 mmol/L fructose and 150
mmol/L sodium citrate) followed by incubation for 30 min at 37ºC.
After incubation, 200_300 spermatozoa were examined
by phase-contrast microscopy at a magnification of 400.
Gametes presenting a clear ballooning of their tail
membranes were counted as swollen. Subclasses of swelling
was not assessed.
2.6 Electron microscopy
Sperm samples were centrifuged at 2 000 ×
g for 10 min and the supernatant was discarded. The sperm pellet
was fixed in cold Karnovsky fixative reagent (containing
2.5% glutaral dehyde, 2% paraformaldehyde in a 0.1
mol/L [pH 7.3] sodium cacodylated buffer) and maintained at
4ºC for 2 h. Fixed spermatozoa was washed in 0.1
mol/L cacodylate buffer (pH 7.2) for 12 h, postfixed in 1%
buffered osmium tetroxide for 1 h at 4ºC and dehydrated
and embedded in Epon Araldite. Ultrathin sections were
cut with a Supernova ultramicrotome (Reickert Jung,
Vienna, Austria), mounted on copper grids, stained with
2% uranyl acetate in 50% methanol for 10 min, followed
by 1% lead citrate for 7 min and then observed and
photographed with a Philips CM10 transmission electron
microscope (TEM; Philips Scientifics, Eindhoven, The
Netherlands). For each patient, 300 ultrathin sperm
sections were analyzed.
2.7 Statistical analysis
χ2 or Fisher's exact test were used to compare the
extent of FSH autoantibody presence among the different
groups. Coefficients of correlation were calculated by
Spearman's correlation analysis. All hypothesis tests
were two-tailed with statistical significance assessed at
the P-value < 0.05 level. The data are expressed as the
mean ± SEM. Statistical analysis was conducted using
SPSS 11.5 for Windows software (SPSS, Chicago, IL,
USA).
3 Results
3.1 The precision of the ELISA method to detect
anti-FSH antibody
The samples with high, middle and lower optical
density were selected and each sample was detected 12 times
at the same time. Each sample was repeated this
experiment for 10 days and the detection result was analyzed
to obtain the coefficient of variation (CV). The results
showed that CV is less than 7% for both intra- and
inter-variation (Table 1).
3.2 The specificity of the ELISA method to detect
anti-FSH antibody
Positive serum from one patient (200 μL, 1:500 dilutions) was selected specifically and cultured with 20
ng, 10 ng and 5 ng FSH at 37ºC for 1 h and then the mixture
was centrifuged at 5 000 × g for 15 min at room
temperature to remove the immuno-complex. The
supernatant (100 μL) was added to FSH coated 96-well microtiter
plates and detected in duplicate by ELISA. The
absorbance was compared between the pre-cultured and
post-cultured samples. Another assay was performed after
samples were cultured with LH. The absorbance was
significantly lower after the samples were cultured with
20 ng, 10 ng, 5 ng FSH compared with the samples that
were not cultured with FSH. Although culturing with
different concentrations of LH also decreased the absorbance, there was no significant difference (Figure
1).
3.3 Detection of anti-FSH antibody in clinical samples
The serum from both the patients and control groups
were assayed for the presence of anti-FSH antibody by
the ELISA method described above. The percentage of
anti-FSH antibody-positive patients with
oligozoospermia and asthenozoospermia was 22.4% (22/98), which
was significantly higher than that in fertile subjects (4%,
2/50, P < 0.05) and than that in infertile patients with
normal sperm concentration and motility (6.7%, 2/30,
P < 0.05). However, it was significantly lower than that
in the patients with obstructive azoospermia (54.5%,
12/22, P < 0.05). Furthermore, the concentration of anti-FSH
antibody represented by absorbance in the patients with
azoospermia was significantly higher than that in
oligozoospermia and/or asthenozoospermia (0.51 ± 0.10
vs. 0.27 ± 0.11) (P < 0.05), infertile people with normal
sperm concentration and motility (0.51 ± 0.10
vs. 0.21 ± 0.12) (P < 0.05) and fertile people (0.51 ± 0.10
vs. 0.19 ± 0.07) (P < 0.05) (Table 2).
3.4 Effects of FSH autoantibody on the percentage of
HOS cells
Forty-four patients with oligozoospermia and asthenozoospermia were selected. Twenty-two of the
patients were positive for FSH-autoantibody and the
remaining 20 patients were negative. There was a
statistically significant increase in the percentage of functional
membrane damage to spermatozoa in the FSH
antibody-positive group in comparison with the values in the
control group (Figure 2). The average percentage of
HOS-positive spermatozoa (swollen) was 45.1% in the FSH
antibody-positive group and 59.1% in the FSH
antibody-negative control group. This difference was statistically
significant (Figure 3).
3.5 Characterization of sperm ultrastructure
We further characterized the ultrastructure of the
spermatozoa from the 44 samples described above. TEM
micrographs of longitudinal and cross-sections of
spermatozoa showed that those in the negative group were
characterized by regular nuclei with condensed
chromatin (Figure 4A). For the spermatozoa in the positive group,
the outer acrosomal membrane was located far from the
nucleus and detachment of the acrosome was observed
(Figure 4B). The axonemes showed normal structure
with visible dynein arms (Figure 4A, B).
4 Discussion
Mammalian spermatogenesis is a finely tuned and
complex process involving intimate interactions among
cells in the two compartments of the testis. In this highly
organized event, the development of an undifferentiated
diploid germ cell into a fully differentiated and mature
spermatozoon is orchestrated in a period unique for each
species. FSH, which is one of the two glycoprotein
hormones produced by the pituitary gland in response to the
stimulus from the hypothalamic GnRH [11], is involved
in the process. Understanding the mechanisms by which
FSH regulates spermatogenesis has become a focus for
investigators working in the area of male reproductive
endocrinology, infertility and contraception. The critical
role of FSH in maintaining qualitative and quantitative
spermatogenesis has also been a topic of debate, with
different interpretations depending on the species [12].
A large body of evidence suggests that FSH is required
for primate spermatogenesis [13]. However, there are
different views about the role of FSH in regulating
spermatogenesis in rodents, particularly in the adult [14]. The
role of anti-FSH antibody in regulating spermatogenesis
in humans is still unknown. We established an ELISA
method to detect anti-FSH antibody using purified FSH
and specific peptides as antigens. This ELISA assay
showed tight inter- and intra-variation. Meanwhile, we
used purified FSH and LH to perform an immune neutralization assay. Even though FSH and LH share a
common α-subunit, the absorbance neutralized by LH was
little decreased, suggesting that the method possesses
good precision and specificity.
Because FSH shares the common glycoprotein alpha
subunit with LH, human chorionic gonadotrophin and
thyroid-stimulating hormone etc., results will be positive
if the antibody to FSH is reactive to the common
alpha-subunit. For this reason, the experiment was conducted
twice using purified FSH and peptide specific to
beta-subunit as antigens to obtain a positive result specific to
the anti-FSH antibody. It is worth noting that the
presence of antibodies might interfere with FSH assays. We
are trying to find a way to exclude this effect, e.g.,
separating the antibody from the immune compound before
measuring the concentration of FSH.
Reimand et al. [9] hypothesized that production of
autoantibody might be initiated by the inflammation
induced by viral and bacterial infection because there is no
evidence of ovary tissue directed autoantibodies being
detected in their patients. It is unclear whether the
production of anti-FSH antibody is developed as a
consequence of immune system activation by specific auto
antigens or by viral and bacterial inflammation. Our
laboratory is currently analyzing the epitope of FSH purified
from the patients with anti-FSH antibody to investigate
the mechanism of anti-FSH antibody production.
The results of ELISA detection showed that the
prevalence of anti-FSH antibody in the patients with
azoo-spermia, oligozoospermia and asthenozoospermia was
significantly higher than that in the infertile patients with
normal sperm concentration and motility. Moreover, the
level of anti-FSH antibody was higher in the patients with
azoospermia than in other patients. Moudgal et
al. [15] demonstrated that continuous bioneutralization of
endo-genous FSH in bonnet monkeys by specific
immunization produces oligospermia and infertility. The poor quality
of the sperm ejaculated by the FSH-immunized monkeys
was indicated by a variety of parameters including
decreased viability, motility and gel penetrability, as well as
acrosin and hyaluronidase activities [16]. Therefore, it
is possible that the anti-FSH antibody could decrease the
biological activity of serum FSH leading to the poor
support of spermatogenesis process by the Sertoli cells.
In the present study, injury to the plasma membrane
in the tail region of spermatozoa, as assessed by the HOS
test, correlated significantly with the presence of FSH
autoantibody. Furthermore, damaged acrosomal membranes, analyzed by TEM, were found to be
associated with the presence of FSH autoantibodies. However,
the mechanisms that caused the damage of membranes
should be further explored.
Acknowledgment
This work was supported partly by Natural Scientific Foundation from Jiangsu Province, China
(BK2006135).
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