Download INTERFERON a-2B MAY IMPAIR TESTICULAR HISTOLOGY

Archives of Andrology, 50:379–385, 2004
Copyright # Taylor & Francis Inc.
ISSN: 0148-5016 print / 1521-0375 online
DOI: 10.1080/01485010490474823
INTERFERON a-2B MAY IMPAIR TESTICULAR
HISTOLOGY INCLUDING SPERMATOGENESIS
IN A RAT MODEL
E. ULUSOY, S. C° AYAN, N. YILMAZ, S. AKTAS° , D. ACAR, and
E. DORUK
Department of Urology, University of Mersin School of Medicine, Mersin, Turkey
Interferon-a has been used in various diseases at the reproductive ages. However, the effect of
interferon-a on testicular histology has not been studied in literature. The aim of this study was
to investigate the effects of interferon a-2B on testicular histology including spermatogenesis in a
rat model. Seventeen adult male Wistar albino rats were divided into 3 groups: Six rats received
7.500 units (5 MIU=m2) of interferon a-2B (Intron), considered clinical treatment dose range. Six
rats received 30.000 units (20 MIU=m2) of interferon a-2B (Intron), considered high treatment dose.
Five rats served as a control group receiving 0.5 mL of saline injection. All injections were done
intraperitoneally 3 times weekly for 3 weeks under inhalation anesthesia. All rats underwent bilateral orchiectomy 30 days after the experiment. Histological examination included the mean seminiferous tubular diameter (STD), germinal epithelial cell thickness (GECT), and testicular biopsy
score (TBS). The mean STD was significantly lower in the low-dose interferon and high-dose interferon groups than in the control group (p ¼ 0.008 and p ¼ 0.004, respectively). The mean GECT
was significantly lower in the low-dose interferon and high-dose interferon groups than in the control group (p ¼ 0.008 and p ¼ 0.004, respectively). The mean TBS was significantly lower in the lowdose interferon group (p ¼ 0.05) and the high-dose interferon group (p ¼ 0.01) than in the control
group. The decreases in the mean values of the STD, GECT and TBS were not related to the interferon dose. Interferon a-2B may impair testicular histology even in clinical widely used treatment
dose. Therefore, men at the reproductive ages should be fully informed for the use of interferon-a in
the treatment of various diseases.
Keywords
interferon, rat, spermatogenesis, testis
INTRODUCTION
Interferons are widely used in the treatment of various diseases including acute and
chronic viral illnesses and certain autoimmune and neoplastic diseases [8]. These agents
have a variety of biologic roles, including antiviral activity, regulation of cell growth, and
modulation of cellular immunity. Interferon-a is the most commonly used antiviral drug
among the interferons as having in vitro antiviral and biologic effects on human cells [6].
Address correspondence to Ercüment Ulusoy, M.D., Associate Professor of Urology, University of Mersin
School of Medicine, Department of Urology, Zeytinlibahc° e Caddesi, 33079-Mersin, Turkey. E-mail: erulusoy@
hotmail.com
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The use of interferons is associated with early side effects such as a flu-like syndrome
and later consequences of treatment which include hematologic, infectious, autoimmune,
neuro-psychiatric, and hormonal problems [17]. Interferon-a has been used in many male
and female patients at the reproductive ages. Interferon-a has been shown to impair
fertility and alter hormone levels in females [13, 15]. However, the effect of interferon-a on
testicular function in males undergoing interferon treatment has been controversial in
literature.
The aim of this study was to investigate the effects of interferon-a on testicular histology including spermatogenesis in an animal model.
MATERIALS AND METHODS
Study Design
The study included 17 adult male Wistar albino rats weighing between 240 and 280 gm,
and all rats were maintained on a 12L: 12D cycle. The experiments were approved by the
ethical committee on animal research at our institution. We provided appropriate care and
use of the laboratory animals as recommended by the Board of Registry publication
guidelines.
The rats were divided into 3 groups: 6 rats received 7.500 units (5 MIU=m2) of interferon a-2B (Intron), considered in clinical treatment dose range, 3 times weekly for 3
weeks (low-dose interferon group). Six rats received 30.000 units (20 MIU=m2) of
interferon a-2B (Intron), considered high treatment dose, 3 times weekly for 3 weeks (highdose interferon group). Five rats served as a control group, receiving 0.5 mL of saline
injection 3 times weekly for 3 weeks. All injections were done intraperitoneally under
inhalation anesthesia. After 30 days, bilateral orchiectomy was performed through standard ilioinguinal incisions, and the rats were sacrified by pentobarbital overdose
(200 mg=kg) and bilateral thoracotomy. Histological examination included the mean
seminiferous tubular diamater (STD), germinal epithelial cell thickness (GECT), and
testicular biopsy score (TBS).
Histological Preparation and Evaluation
The testicles were fixed in Bouin’s solution (7.5 ml of saturated picric acid, 2.65 ml of
glacial acetic acid and 2.5 ml of 7% formaldehyde), postfixed in 70% alcohol and
embedded in paraffin blocks. Five mm sections were obtained, deparaffinized, and stained
with Hematoxylin and Eosin to examine the mean STD, GECT, and TBS.
Testicular tissue was evaluated with standard light microscopy by a blinded observer in
random order. Three slides prepared from the upper, lower and mid portions of the testes
were evaluated completely for each testis. The mean STD, GECT, and TBS were used to
evaluate in 20 seminiferous tubule of each section. The mean STD and GECT were calculated using a microscope-adaptable micrometer. Therefore, the mean STD and GECT
of each testis were determined in mm. The mean TBS was graded using modified Johnsen’s
score [20]. A score of 0–12 was given to each tubule according to epithelial maturation.
This method of histologic analysis gives an accurate measurement of the degree to which
Interferon a-2B and Testicular Histology
381
spermatozoal maturation is taking place within the seminiferous tubule and also the level
of spermiogenesis.
Statistical Analysis
All data were expressed as mean standard deviation (SD). Multiple comparisons
were made using Mann Whitney U test between the groups. A p value of <0.05 was
considered as statistically significant.
RESULTS
Figures 1, 2, and 3 show histology of the testis in a rat of the low-dose interferon group,
high-dose interferon group, and the control group, respectively. The mean values of the
STD, GECT and TBS (modified Johnsen’s score) in all groups are shown in Table 1. The
mean STD was significantly lower in the low-dose interferon and high-dose interferon
groups than in the control group (p ¼ 0.008 and p ¼ 0.004, respectively). The mean GECT
was significantly lower in the low-dose interferon and high-dose interferon groups than in
the control group (p ¼ 0.008 and p ¼ 0.004, respectively). The mean testicular biopsy score
(modified Johnsen’s score) was lower in the low-dose interferon and high-dose interferon
groups than in the control group, revealing statistical significance (p ¼ 0.05 and p ¼ 0.01).
Figure 1. Histology of the testis in a rat of the low-dose interferon treatment group, showing decreases in seminiferous tubular diameter, germinal epithelial cell thickness and testicular biopsy
score.
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E. Ulusoy et al.
Figure 2. Histology of the testis in a rat of the high-dose interferon treatment group, showing decreases in seminiferous tubular diameter, germinal epithelial cell thickness and testicular biopsy
score.
The mean values of the STD, GECT and TBS (modified Johnsen’s score) were not
statistically significantly different between the low-dose interferon and high-dose
interferon groups (p ¼ 0.429, p ¼ 0.792, and p ¼ 0.931, respectively), showing that the
decreases in the mean values of the STD, GECT, and TBS were not related to the dose of
the interferon treatment.
DISCUSSION
Interferon-a is approved in the United States for treatment of condyloma acuminatum,
chronic hepatitis C, chronic hepatitis B, Kaposi’s sarcoma in HIV-infected patients, and
other malignancies including leukemia, multiple myeloma, non-Hodgkin’s lymphoma, and
urogenital carcinomas such as renal cell cancer, bladder cancer, and ovarian cancer [8].
Interferons may have direct effects such as inhibition of cell proliferation, induction of
differentiation and antiviral effects, and indirect effects such as stimulation of immunologic functions, anti-angiogenesis, and production of other cytokines [1].
Human interferons consist of the multiple type 1 species (alpha, beta, omega and
kappa), and the one type 2 species (gamma) [1]. However, three types of human interferons
(a, b, w) have been recognized in the clinical use [8]. Interferon w has less antiviral
activity but more potent immunoregulatory effects. The principal antiviral interferons,
Interferon a-2B and Testicular Histology
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Figure 3. Histology of the testis in a rat of the control group, showing normal seminiferous tubular
diameter, germinal epithelial cell thickness and spermatogenesis.
interferon-a, and interferon-b are approximately 30% homologous at the amino acid level.
In addition, interferon a is actually a family of multiple species that share a high degree of
amino acid sequence homology (>70%) but differ in vitro antiviral and biologic effects on
human cells [6]. Therefore, interferon-a is a commonly used antiviral drug among the
interferons. The beneficial effect of interferon-a in the treatment of viral hepatitis is related
to the activation of lymphocytes such as T cells and natural killer cells, which participate
in the elimination of infected cells.
The side effects of interferon-a during treatment are seen as general symptoms with a
flu-like syndrome in 40 to 85% of cases, neuropsychiatric signs in up to 30% of cases,
Table 1. The mean values of the STD, GECT, and TBS (modified Johnsen’s score) of the groups
Groups
Low-dose interferona (n ¼ 6)
High-dose interferonb (n ¼ 6)
Control (n ¼ 5)
a
Seminiferous tubular
diameter SD (mm)
Germinal epithelial
cell thickness SD (mm)
Testicular biopsy
score SD
219.8 16.43
226.7 14.04
275.5 4.03
62.7 5.33
62.66 4.51
81.8 5
11.76 0.22
11.83 0.06
11.95 0.05
versus control p ¼ 0.008 for STD, p ¼ 0.008 for GECT and p ¼ 0.05 for TBS.
versus control p ¼ 0.004 for STD, p ¼ 0.004 for GECT and p ¼ 0.01 for TBS.
b
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thyroid dysfunction in 5 to 10% of cases, hematological side effects in more than 50% of
cases, dermatological abnormalities frequently, and cardiovascular and ophthalmic
disorders rarely [7].
Although interferon-a has been used in the treatment of mumps orchitis, the preventive
role of the interferon treatment has still been controversial. Significant preventive effects of
systemic treatment with interferon a-2B for infertility from mumps orchitis has been
reported by two articles [5, 11]. In contrast to these studies, Yeniyol et al. reported the role
of interferon-a for the prevention of testicular atrophy in postpubertal men with unilateral
mumps orchitis [19]. They found that systemic treatment with interferon a-2B was not
completely effective in preventing testicular atrophy after mumps orchitis.
Deterioration of reproductive function and alteration of serum hormone have been
shown with the use of interferon-a in women [13, 15]. Interferons have been shown to
inhibit Leydig cell steroidogenesis in vitro, through inhibition of StAR expression [12, 16].
Corssmit et al. reported acute effects of interferon-a treatment on the hypothalamicpituitary-testicular axis in healthy men, and they found that interferon treatment
decreased serum testosterone and free androgen index [3]. Therefore, interferon treatment
may change feedback relationships between the pituitary and the testis. Karamfilov et al.
investigated the effect of low dose interferon-a therapy in patients with melanoma, and
they found no significant effect on serum inhibin B level as a marker of male fertility with
the use of low dose interferon-a [10]. However, the effect of interferon-a on spermatogenesis has not been clearly demonstrated in men with normal testes.
Possible effects of interferon-a on sperm parameters in the treatment of male infertility
have been controversial. Yamamoto and Miyake reported successful use of interferon for
male infertility [18]. However, azoospermia was reported in a patient receiving interferon
alpha for a stage 3 melanoma [14]. Hibi et al. found that daily sperm production and
epididymal sperm concentrations were significantly increased after interferon alpha
treatment in the nude rats [9]. Therefore, they concluded that these promising results with
interferon-a may pave the way for a new approach to treating male infertility. However,
they did not investigate testicular spermatogenesis using Johnsen’s score. In the present
study, we investigated the effects of interferon a-2B on testicular histology including
spermatogenesis in a rat model. We found that the mean STD, GECT, and TBS were
significantly lower in the low-dose interferon and high-dose interferon groups than in the
control group. The decreases in the mean values of the STD, GECT and TBS were not
related to the interferon dose, showing that interferon might impair testicular histology
regardless of the dose of the treatment.
Interferons have been considered as apoptosis inducing cytokines [2]. Both type 1 and
type 2 interferons have been found in the testis, and are stimulated by viral infections,
particularly in Sertoli and Leydig cells. They are involved in protecting the testis against
viral infections [4]. It has been demonstrated that interferons induce apoptosis by
activating the death receptor cascade in cancer patients [2]. However, testicular apoptosis
has not been studied with the use of interferons. Increased apoptosis might impair
spermatogenesis in male patients undergoing interferon treatment.
In conclusion, our findings suggest that interferon a-2B may impair testicular histology
even in clinical widely used treatment dose. Therefore, men at the reproductive ages should
be fully informed for the use of interferon-a in the treatment of various diseases.
Interferon a-2B and Testicular Histology
385
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