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- Original Article -
Sperm quality improvement after natural anti-oxidant treatment of asthenoteratospermic men with leukocytospermia
Paola Piomboni1,2, Laura
Morgante2,3, Vincenzo De Leo2,3
1Department of Surgery, Biology Section, 3Department of Pediatrics, Obstetrics and Reproductive Medicine, Gynecology
and Obstetrics Section, University of Siena, Siena 53100, Italy
2Center for Diagnosis and Treatment of Couple Sterility, Siena Hospital, Siena 53100, Italy
Aim: To study the immune-modulating and anti-oxidant effects of beta-glucan, papaya, lactoferrin, and vitamins C
and E on sperm characteristics of patients with asthenoteratozoospermia associated with leucocytosis.
Methods: Fifty-one patients referred to our Sterility Center for semen analysis were selected. Sperm parameters were assessed
before and after patient's treatment with beta-glucan, lactoferrin, papaya, and vitamins C and E. DNA damage was
assessed by the acridine orange test and sperm structural characteristics were evaluated by transmission electron
microscopy. Results: After 90 days of treatment, an increase in the percentage of morphologically normal sperm
(17.0 ± 5.2 vs.
29.8 ± 6.5) and total progressive motility
(19.0 ± 7.8 vs.
34.8 ± 6.8) were detected. Structural sperm
characteristics as well as chromatin integrity were also improved after treatment. In terms of leukocyte concentration
in seminal fluid, a significant reduction was recorded
(2.2 ± 0.9 vs.
0.9 ± 0.2). Conclusion: The treatment of an
inflammatory process by the synergic action of immune modulators and anti-oxidants could protect sperm during
maturation and migration, leading to improved sperm
function. (Asian J Androl 2008 Mar; 10: 201_206)
Keywords: asthenoteratozoospermia; leukocytospermia; anti-oxidant; beta-glucan; papaya; lactoferrin; vitamin C; vitamin E; chromatin
integrity; transmission electron microscopy
Correspondence to: Prof. Paola Piomboni, Department of Surgery, Biology Section, Policlinico Le Scotte, Viale Bracci, Siena 53100, Italy.
Received 2007-07-27 Accepted 2007-09-17
Many factors can impair male reproductive capacity, causing transient or permanent infertility. In these cases,
leukocytospermia represents an additional risk factor inducing the production of highly toxic reactive oxygen species
(ROS) that impair genital tract accessory glands and sperm cell functions and quality.
High leukocyte counts in seminal fluid are common, even in the absence of inflammatory symptoms, and might be
an indirect sign of viral or microbial infections, past testicle trauma, and varicocele. Although white blood cell lines
play a physiological role in immune surveillance and in eliminating anomalous sperm, many studies indicate that an
increased number of leukocytes in seminal fluid is associated with altered sperm parameters [1_3].
The relationship between inflammation of the genital tract and reduced fertility is unclear. Different stimuli
(chemical, biological, and physical) are known to activate cells of the lymphatic system, inducing an inflammatory
response accompanied by production of ROS . Scrotal varicocele was found to be associated with elevated ROS
levels and decreased levels of anti-oxidants in internal spermatic venous blood compared to peripheral venous
circulation . When the body fails to counteract increased production of these radicals, they might cause oxidative
stress, affecting sperm. Lipid peroxidation causes
structural damage to the acrosome and sperm head and neck,
as well as triggering apoptosis and inducing DNA
breakage . Altered nuclear chromatin structure or DNA
damaged in sperm could be responsible for male infertility. It has been reported that
in vivo fecundity is decreased when more than 30% of sperm show DNA
damage. Moreover, gamete function is impaired by
reduced motility and by inhibition of the acrosome
reaction and fusigenic capacity [6_9].
Anti-oxidant enzymes in seminal fluid therefore play
a fundamental role, replacing the cytoplasmic enzymes
lost by sperm during spermiogenesis. Seminal fluid also
contains non-enzyme anti-oxidants, such as vitamins C
and E, pyruvate, glutathione, and carnitine. Some
authors reported that aging, diet, smoking and lifestyle tend
to reduce immune defenses and anti-oxidant activity,
lowering semen quality and impairing fertilizing capacity [10,
Anti-inflammatory drugs of different types have been
found effective in the treatment of asymptomatic and
symptomatic leukocytospermia [12, 13], and improved
sperm quality has been reported after oral intake of
anti-oxidants [14, 15]. The effect of a therapy combining
the immune-stimulating properties of beta-glucan and
fermented papaya with anti-oxidant vitamins C and E and
the bacteriostatic effect of lactoferrin has never been
assessed in asthenospermic males. Beta-glucan, a polysaccharide extracted from yeast cell walls, is known
for its immune-stimulating properties. It reinforces natural
defenses against viral infections, bacteria, fungi,
parasites and neoplastic cells, and stimulates tissue repair [16,
17]. Many recent studies have shown the efficacy of
papaya against peroxidation and the genotoxicity of free
radicals [18, 19]. The anti-oxidant properties of fresh
papaya, which contains vitamins and amino acids, are
increased by fermentation, improving its
immune-modulating activity . Lactoferrin, a protein found in milk
and other products of the exocrine glands of the
digestive, respiratory, and reproductive systems, is a natural
anti-oxidant and a powerful activator of natural killer cells. It
modulates the migration, maturation, and function of cells
of the immune system . Ascorbic acid and
alpha-tocopherol, vitamins C and E, respectively, are normally
present in seminal fluid where they counteract
peroxidation by virtue of their anti-oxidant properties. Their
concentration in seminal fluid is correlated with daily
The aim of the present study was to assess the
synergic effect of the immune-modulating and anti-oxidant
properties of beta-glucan, papaya, lactoferrin, and
vitamins C and E on sperm quality in men with
asthenoteratozoospermia and leukocytospermia.
2 Materials and methods
We selected 36 patients referred to our Sterility
Center for semen analysis after 12_18 months of sexual
intercourse without conception. The patients had
leukocyte counts in seminal fluid exceeding
1 × 106. Azoospermic men were excluded. The patients enrolled in this
study did not have a history of endocrine or anatomical
The diagnosis of leukocytospermia associated with
asthenoteratozoospermia was confirmed by a second spermiogram 1 month after the first. In all patients,
microbiological screening of seminal fluid and urine for
common microorganisms Mycoplasma, Trichomonas
vaginalis and Chlamydia trachomatis gave negative
results. The mean age of patients was 32 years (range,
As controls we selected 15 men (mean age 30 years;
range, 25_41 years) with the same seminal characteristics,
associated leukocytospermia and negative sperm culture,
for whom no therapy was prescribed.
The patients were treated for 3 months with two
tablets per day of a formula (Fattore M). Each tablet
contained 20 mg beta-glucan, 50 mg fermented papaya,
97 mg lactoferrin, 30 mg vitamin C, and 5 mg vitamin E
(Progine, Florence, Italy).
2.4 Analysis of seminal fluid
Before the spermiogram, patients observed 4 days
of sexual abstinence. Samples were assessed according
to World Health Organization (WHO) guidelines ,
using an inverted phase-contrast microscope with Hoffmann lens (Olympus, Milan, Italy) and platform
heated to 37ºC. Eosin Y was used to detect necrotic
sperm. Morphological examination of the specimens was
carried out after staining with modified
Papanicolaou reagent for sperm. One hundred sperm were counted in
each sample and sperm organelle morphological parameters (nucleus, acrosomal and postacrosomal regions,
and flagellum) were evaluated by WHO criteria . All
patients repeated the spermiogram after 3 months.
2.5 Peroxidase staining
Leucocytes were counted after peroxidase staining.
Briefly, 0.0375% H2O2 was added to 4 mL benzidine stock
solution (0.0125% w/v benzidine [Sigma Aldrich, Milan,
Italy], in 50% ethanol). Ten microliters of ejaculate was
mixed with 20 μL fresh
benzidine-H2O2 solution. After
5 min, 160 μL phosphate-buffered saline was added and
peroxidase-positive (round brown cells) and
peroxidase-negative (unstained) cells counted using a Makler
chamber (Sefi-Medical Instrumets, Haifa, Israel) and
phase-contrast microscope (Olympus, Milan, Italy).
2.6 Acridine orange (AO) staining
Sperm chromatin DNA was assessed by AO (Sigma Chemical, St. Louis, MO, USA) fluorescence method
described by Tejada et al. . Briefly, after air-drying,
the sperm smears were fixed in Carnoy's solution (methanol-glacial acetic acid 3:1) overnight at 4ºC. After
they were air-dried, the samples were stained with AO
solution for 5 min. The AO staining solution was
prepared daily by adding 10 mL of 1% AO stock solution in
distilled water to a mixture of 40 mL of 0.1 mol/L citric
acid and 2.5 mL of 0.3 mol/L
Na2HPO47H2O, pH 2.5.
Then the samples were rinsed and mounted with distilled
water. The percentage of sperm with normal
double-stranded DNA (green) was determined by randomly
scoring 100 sperm under a fluorescence microscope (DMRB;
Leica, Lodi, Italy) with × 400 magnification and
excitation of 450 nm_490 nm. Red or yellow fluorescent
sperm indicated denatured or single-stranded DNA.
2.7 Transmission electron microscopy (TEM)
Aliquots of 19 semen samples, before and after treatment, and 10 samples before and after 3 months
without any therapy were examined by TEM. Sperm samples were fixed in Karnovsky's reagent, rinsed
overnight in 0.1 mol/L cacodylate buffer (pH 7.2), postfixed
in 1% buffered OsO4, dehydrated, and embedded in
Epon-Araldite (Fluka, Milan, Italy). Ultrathin sections were
cut with an LKB ultramicrotome (Vienna, Austria) and
stained with uranyl acetate and lead citrate.
Observations of 100 sperm sections were made with a TEM CM
10 (Philips, Eindhoven, the Netherlands), at
magnifications of × 5 000 to × 75 000, by two highly trained
evaluators who did not know the identity of the samples.
2.8 Statistical analysis
The data were collected and analyzed using the
commercial software GraphPad Prism4 (GraphPad Software,
San Diego, CA, USA). Results were expressed as mean ± SD. Standardized skewness and kurtosis values
were used to determine the normal distribution of data.
The Mann-Whitney U-test was used for comparisons.
Statistical significance was set at P < 0.05.
The first spermiogram obtained before therapy showed asthenoteratozoospermia and leukocytospermia;
the diagnosis was confirmed by the second spermiogram
carried out 1 month later. Mean sperm count,
progressive motility, and normal sperm morphology were below
the normal range in all patients (Table 1). Staining with
peroxidase revealed a high mean leukocyte count. After
the end of therapy, there was no significant change in
sperm count, but morphology and motility improved, and
leukocyte count significantly decreased.
Light microscopy examination of sperm showed
diffuse sperm anomalies in patients before therapy. The
acrosome was frequently absent or abnormally shaped
and the nucleus was frequently malformed. Sperm tails
were twisted, disrupted, or altered. All of these tail
anomalies could explain the motility reduction. After
therapy, a general improvement in sperm features was
observed, namely normal-shaped nucleus, acrosome, tail
structure and extension.
TEM evaluation of basal samples revealed diffuse
structural defects typical of necrosis. The acrosome
was absent or reacted, the chromatin was uncondensed
or disrupted, and the plasma membrane was broken. The
cytoskeletal structures of sperm flagellum were altered
and often rolled up, and the mitochondrial helix was
frequently disorganized with swollen mitochondria
(Figure 1). After therapy, ultrastructural analysis showed that sperm
defects typical of necrosis were reduced compared to
the previous examination (Figure 2).
After therapy, eosin Y staining also confirmed a
significant reduction in sperm with broken plasma
membrane in comparison to basal samples, in which more
than half the sperm had broken plasma membranes (Table 1).
The AO staining for detection of sperm with damaged DNA did not reveal any significant change in the
mean percentage of orange/red abnormal single-stranded
DNA sperm after therapy.
Patients treated with anti-oxidants and beta-glucan
did not complain of any side-effects and all reported feeling
well. Seven men with a history of recurrent respiratory
tract infections contracted fewer infections during
The spermiogram of 15 untreated patients (controls),
carried out 3 months after the first in the absence of any
type of therapy, did not show any significant variation in
sperm number, motility, or morphology (Table 1). The
concentration of leukocytes was almost the same in the
two examinations. Eosin Y staining confirmed the
presence of a higher percentage of dead sperm compared to
WHO parameters. DNA damage detected by AO staining did not show any significant change after 3 months
of interval without any therapy (Table 1).
Asymptomatic inflammatory processes in the testicles associated with leukocytospermia might damage
the seminiferous epithelium, the epididymis, or the
ejaculatory ducts, altering the process of sperm formation and
maturation, as shown by the presence of anomalous sperm [1_3, 23]. A possible mechanism by which
leukocytospermia might alter sperm functions could be
contact of sperm and ROS produced by leukocytes
during co-migration from the seminiferous tubules to the
epididymis. In the sperm cell, one of the main sites of
lipid peroxidation by ROS is the intermediate tail
segment that consists essentially of mitochondria .
Therefore, the reduction in sperm motility observed in
our patients could also be due to altered membrane
permeability with loss of intracellular adenosin triphosphate
(ATP) and axonemal damage. ROS also have a negative
effect on sperm DNA, leading to fragmentation . It
has been suggested that DNA fragmentation could be a
possible cause of increased infertility in males . In
the present study, natural anti-oxidants and beta-glucan
given to patients for 90 days led to improvement in some
sperm parameters, namely motility and morphology, and
reduction in the number of lymphocytes. The
percentage of sperm with DNA damage was also decreased
after anti-oxidant treatment, but without reaching
statistical significance. These results are presumably due to
the double action of the components of the product.
Beta-glucan, fermented papaya, and lactoferrin modulate
immune and inflammatory responses [16_19, 27], whereas
vitamins C and E increase the anti-oxidant defenses of
In men, the combined action of anti-oxidants and
beta-glucan seems to protect sperm structure, reducing
the effects of free radicals. In fact, not only enzymes,
but also anti-oxidant systems, including vitamins C and
E, are fundamental for defense of sperm against
oxidative damage. The effects of the anti-oxidant therapy on
seminal fluid have been the subject of clinical trials that
have shown the individual and synergic action of
vitamins C and E in protecting sperm against oxidative stress
in cases of idiopathic infertility [28, 29]. In some studies,
no effects on conventional semen parameters were found
after vitamin C and E supplementation [30, 31]. Recently,
Ménézo et al.  reported a decrease in DNA
fragmentation but an unexpected increase in sperm
deconden-sation, probably due to the ability of anti-oxidant
vitamins to interfere with interchain disulfide bridges of sperm
protamines. Moreover, sperm DNA damage has been reported by Greco
et al.  to be efficiently treated by vitamins C and E, given orally. Seminal plasma levels of
vitamin C show a positive correlation with the presence
of morphologically normal sperm, probably acting at the
epididymal level . Fermented papaya, moreover, has
strong anti-oxidant properties, eliminating free radicals
and increasing superoxide dismutase activity [18, 19].
The results of the present study suggest that the tested
formula can be effective in the treatment of certain
spermiogenetic alterations, particularly of the nucleus and
motility apparatus. Improvement in the main sperm
parameters is related to restored cell function sufficient to
enable natural fertilization, or at least less invasive
assisted reproductive techniques. Treatment with natural
supplements containing high concentrations of vitamins
C and E can be considered a valid alternative to
anti-inflammatory drugs in the treatment of asthenospermia
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