Document Type : Erratum
Authors
Abstract
Keywords
The increase in incidences of infertility in men
due to frequent use of a number of therapeutic
drugs has made efforts to study their untoward side
effects on the male reproduction. Various drugs
used for treating diseases are reported to cause
male infertility (
The first nitroimidazole to exert useful clinical activity is metronidazole, (MTZ; 1-[2-hydroxyethyl]2-methyl-5-nitroimidazole), a drug of first choice,
recommended by the clinicians to be consumed
at maximum for seven to ten days for the treatment of
Quantitative studies have indicated marked alterations in the number of germ cells at stage I, V
and XII following intraperitonial administration of
130 mg/kgBW/day of MTZ for seven days in mice
(
From the foregoing it is clearly seen that MTZ at various doses impairs fertility in the males by inhibiting spermatogenic activity and sperm indices. However, a detailed study regarding the effects of therapeutic dose of MTZ for long duration, such as for 4-8 weeks on the male reproductive organs and fertility is still required. Therefore, the aim of the present study is to investigate the effects of the therapeutic and high doses of MTZ on the testis, epididymis, seminal vesicle and fertility as well as on the secretory activities of the latter two organs. For the safety evaluation of the potential effect of the drug on the male reproductive organs, a study with a dose higher than the therapeutic one may be considered in a non clinical trial. The study also deals with the withdrawal effects of high dose of MTZ, 42 days after cessation of the treatment.
In this experimental study, fifty Swiss strain adult (12 weeks old) male mice weighing about 25-30 g were used for the present investigation. The animals were housed under standard laboratory conditions and maintained on pelleted diet and water ad libitum. Approval from the Animal Ethical Committee, Banaras Hindu University, Varanasi, India was obtained for the animal study plan (No. Dean/11-12/CAEC/263).
After recording the initial body weights, all the
animals were divided into five groups of ten each
and treated as follows:
Group I Untreated controls Group II Vehicle-treated controls (distilled water) Group III Administration of MTZ (250 mg/kgBW/
day) for 28 days Group IV Administration of MTZ (500 mg/kgBW/
day) for 28 days Group V Administration of MTZ (500 mg/kgBW/
day) for 28 days followed by sacrificing the animals 42 days after cessation of the treatment.
MTZ (CDH, India) was dissolved in double
distilled water and administered orally. The
human therapeutic dose of MTZ was selected
and translated to mice (
After recording the final body weights the animals were sacrificed by cervical dislocation. Among ten animals from each group, five animals were used for the histological studies and sperm assessment while the other five were used for biochemical studies, fertility test and serum testosterone level. Blood was collected by cardiac puncture to measure the level of serum testosterone. The reproductive organs were dissected out, blotted free of blood and processed for the following studies:
Wet weights of the testis, epididymis and seminal vesicle were recorded to calculate the gonadosomatic index by using the following formula: Gonadosomatic Index (GSI)=(Gonad weight/total body weight) ×100.
Bouin’s fixed testis, epididymis and seminal vesicle were dehydrated and embedded in paraffin. Sections of 5 μm thickness were taken from the mid portion of each testis, all the three regions of epididymis and seminal vesicle, dehydrated in graded series of alcohol and stained with Periodic Acid Schiff reagent followed by counterstaining with Ehrlich’s Hematoxylin.
Frequency of the stages was determined from
one cross section of the testis of the five animals
in each group. All the seminiferous tubules within a cross section of the testis were examined at
×40 and classified according to the stages of the
cycle. The stages of the seminiferous tubules were
classified according to the method of Hess and
Franca (
The diameter of the seminiferous tubules was measured using ocular micrometer at ×40 objective piece.
Concentrations of epididymal sialic acid and
seminal vesicular fructose were estimated using
the methods of Aminoff (
Cauda epididymidis of five mice in each group
was minced thoroughly in the physiological normal saline at 37˚C and used for the assessment of
motility, viability and count according to the WHO
Laboratory Manual (
Evaluation of sperm abnormality was based
on the criteria of Wyrobek and Bruce (
Serum testosterone was measured by ELISA, as described in the instructions provided in the kit (LDN, Germany).
Each male was caged with two proestrus females overnight and according to presence of
vaginal plug and implantation sites in females,
the mating ability and fertility of the males were
assessed respectively. The females were sacrificed by cervical dislocation on the fifteenth day
of cohabitation with males. The ovaries were
removed to count the number of corpus luteum.
To determine the total number of implantation
sites, the dissected out uteri were placed in 10%
ammonium sulfide solution, which stained the
hemosiderin pigment of resorbed implanted
sites blue-black (
Corpus luteum – [number of resorbed implants + number of live implants + number of dead implants] Postimplantation loss was equal to the total number of resorbed and dead implants.
All the data were analyzed statistically by one way ANOVA followed by Newman-Keul’s test.
Body weight and number of live implants as well as pre- and postimplantation loss were analyzed using Student’s t test. Values were considered significant at p<0.05.
No significant differences were found between
the initial and the final body weights of the MTZtreated mice and the controls at therapeutic and
high dose (
Administration of MTZ at the therapeutic
dose did not induce significant changes in the
weights of the testis and epididymis while the
drug at the high dose resulted in significant reductions in the weight of these organs as compared with the controls. Forty two days after
cessation of the treatment, weight of the organs
recovered to the control values. Administration
of MTZ at any dose did not induce significant
reduction in the weight of the seminal vesicle
compared with that of controls (
Effect of the oral administration of MTZ on body weight and weight of testis, epididymis and seminal vesicle (values are mean ± SE of five animals)
Groups | Body weight (g) | Weight of the reproductive organs (mg/100 gBW) | |||
---|---|---|---|---|---|
Initial BW | Final BW | Testis | Epididymis | Seminal vesicle | |
23.2 ± 1.35 | 28.0 ± 1.52 | 316.87 ± 20.6 | 131.36 ± 9.52 | 230.79 ± 22.41 | |
23.8 ± 0.19 | 26.8 ± 0.58 | 310.40 ± 21.17 | 134.21 ± 6.15 | 230.84 ± 27.1 | |
25.8 ± 0.48 | 28.4 ± 0.51 | 297.97 ± 9.09 | 122.54 ± 9.28 | 200.05 ± 20.11 | |
23.4 ± 1.32 | 27.8 ± 0.79 | 189.96 ± 4.95a | 101.23 ± 5.3a | 169.45 ± 14.53 | |
27.6 ± 0.51 | 33.2 ± 0.86 | 291.31 ± 9.94b | 166.63 ± 8.29b | 245.97 ± 25.57 | |
*; Administration of MTZ for 28 days followed by sacrificing the animals 42 days after cessation of the treatment, a; As com- pared to Groups I and II: p< 0.05 and b; As compared to Group IV: p< 0.05.
The testis of untreated and vehicle-treated
controls (
(A-D) Transverse section (T.S.) of the Testis of control (A) showing normal appearance of seminiferous tubules. (B-D) MTZ (500 mg/kgBW/day)-treated mouse for 28 days where (B) shows the shrinkage of the seminiferous tubules, depletion, disorganization, vacuolization and sloughing of the germ cells and appearance of multinucleated giant cells in the seminiferous tubules; (C) shows the giant cell (arrow); (D) shows the recovery in spermatogenesis in animals sacrificed 42 days after cessation of the treatment.
Quantitative analysis of the spermatogenic cycle
revealed no alterations in all the stages of the seminiferous tubules in the testis of mice administered with
therapeutic dose of MTZ as compared with the controls. In contrast, a significant decrease was observed
in stages I-VIII after high dose of MTZ-treatment as
compared with the controls (
Therapeutic dose of MTZ treatment did not induce any alteration in the diameter of the seminiferous tubules while a significant decrease in the
same was noted in the testis of mice administered
with high dose of the drug as compared with controls (
Effect of oral administration of MTZ on the percentage frequencies of stages of the spermatogenic cycle (values are mean ± SE of five animals)
Groups | Weight of the reproductive organs (mg/100 gBW) | ||||
---|---|---|---|---|---|
Stage I-IV | Stage V-VI | Stage VII-VIII | Stage IX-X | Stage XI-XII | |
28.05 ± 1.56 | 17.80 ± 3.29 | 27.37 ± 1.22 | 13.20 ± 1.73 | 15.53 ± 2.13 | |
25.32 ± 1.56 | 23.18 ± 2.5 | 25.19 ± 3.87 | 11.99 ± 2.02 | 17.67 ± 4.05 | |
25.64 ± 1.97 | 21.61 ± 0.9 | 25.98 ± 1.43 | 11.64 ± 1.09 | 15.09 ± 1.65 | |
IV. MTZ (500 mg/kgBW/day) | 06.85 ± 5.88 a | 12.91 ± 7.09 a | 05.99 ± 2.46 a | 36.93 ± 9.5 a | 37.27 ± 14.92 |
V. MTZ (500 mg/kgBW/day)* | 23.64 ± 1.85 b | 23.05 ± 1.26 b | 24.43 ± 1.26 b | 10.69 ± 0.74 b | 18.14 ± 1.73 |
*; Administration of MTZ for 28 days followed by sacrificing the animals 42 days after cessation of the treatment, a; As com- pared to Group I and II: p<0.05 and b; As compared to Group IV: p< 0.05.
Effect of oral administration of MTZ on the diameter and number of various types of germ cells of stage VII of the seminiferous tubules (values are mean ± SE of five animals)
Groups | Type A | Preleptotene | Pachytene | Round | ||
---|---|---|---|---|---|---|
Diameter (μm) | spermatogonia | spermatocytes | spermatocytes | spermatids | ||
215.55 ± 06.80 | 1.92 ± 0.19 | 51.32 ± 2.19 | 71.32 ± 4.22 | 175.00 ±14.25 | ||
223.28 ± 09.90 | 2.16 ± 0.31 | 49.44 ± 5.88 | 75.00 ± 8.08 | 174.34 ±18.90 | ||
218.96 ± 07.84 | 1.92 ± 0.30 | 40.36 ± 1.55 | 56.96 ± 3.36 | 147.92 ± 06.53 | ||
179.71 ±11.20 a | 0.60 ± 0.60 a | 09.68 ± 9.60 a | 11.60 ± 11.6 a | 024.48 ± 24.40 a | ||
198.72 ± 09.34 | 1.6 ± 0.28 b | 47.36 ± 5.13 b | 79.04 ± 7.49 b | 159.36 ± 20.69 b | ||
*; Administration of MTZ for 28 days followed by sacrificing the animals 42 days after withdrawal of the treatment, a; As compared to Groups I and II: p< 0.05 and b; As compared to Group IV: p< 0.05.
The epididymis of the untreated and vehicletreated controls exhibited normal histological
features. In the Swiss mice, five segments (I-V)
were noticed in the epididymis. Segments I-III
constituted the caput (
T.S. of various segments of the epididymis. (A-E) Segments of I-V of control to show normal histological features. (FJ) Segments of I-V of MTZ (500 mg/kgBW/day)-treated mouse for 28 days showing PAS-positive material, sperm debris and sloughed off germ cells in the lumina of segments II (Fig G), IV (Fig I) and V (Fig J).
Treatment with MTZ at any dose did not cause
any alteration in the histoarchitecture of the seminal vesicle as compared with the control (
Administration of MTZ at any dose did not induce significant alterations in the concentrations
of sialic acid in the epididymis and fructose in the
seminal vesicle (
Therapeutic dose of MTZ did not cause significant reductions in the motility, viability and count
of epididymal spermatozoa. By contrast these
sperm indices declined significantly in mice administered with high dose of MTZ. Percentage of
abnormal spermatozoa increased in MTZ-treated
groups, though, the values were not significant.
Withdrawal of the treatment, however, resulted in
marked recovery in motility, viability and count of
spermatozoa in the epididymis comparable to that
of control (
T.S. of the Seminal vesicles of control (A) to show normal histological features (B) MTZ (500 mg/kgBW/day)-treated mouse for 28 days showing unaltered histology.
Effect of the oral administration of MTZ on the concentrations of sialic acid in the epididymis and fructose in the seminal vesicle (values are mean ± SE of five animals)
Groups | Concentration of sialic acid (μmole/100 mg of tissue) | Concentration of fructose (μg/100 mg of tissue) |
---|---|---|
196.91 ± 46.08 | 264.53 ±15.4 | |
195.71 ± 18.3 | 259.41 ±15.18 | |
212.88 ± 41.26 | 237.75 ±13.78 | |
155.51 ± 22.55 | 235.09 ± 21.02 | |
214.21 ± 8.91 | 243.80 ± 15.73 | |
*; Administration of MTZ for 28 days followed by sacrificing the animals 42 days after cessation of the treatment.
Effect of the oral administration of MTZ on sperm motility, viability, morphology and count in the cauda epididymidis (values are mean ± SE of five animals)
Groups | Motility (%) | Viability (%) | Abnormal morphology (%) | Count (X 106) |
---|---|---|---|---|
64.83 ± 2.69 | 63.25 ± 4 | 38.80 ± 5.47 | 13.98 ± 2.6 | |
67.80 ± 6.82 | 64.95 ± 1.79 | 35.73 ± 5.43 | 13.66 ± 2.19 | |
47.60 ± 4.76 | 53.67 ± 4.67 | 51.65 ± 5.59 | 09.84 ± 1.73 | |
28.23 ± 8.40 a | 23.41 ± 2.94 a | 62.40 ± 9.72 | 02.19 ± 0.34 a | |
65.78 ± 1.03 b | 64.76 ± 2.04 b | 42.73 ± 5.97 | 11.54 ± 1.4 b | |
*; Administration of MTZ for 28 days followed by sacrificing the animals 42 days after cessation of the treatment, a; As com- pared to Groups I and II: p<0.05 and b; As compared to Group IV: p<0.05.
No significant change was found in the level of
serum testosterone caused either by therapeutic or
high dose of MTZ as compared with the control
(
Mating ability of all the treated males remained almost unaffected comparable to that of
the controls. Marked reduction was noted in the
fertility of the males treated with high dose of
MTZ; 67% of treated males became infertile after the treatment. Fertility of the virgin females
impregnated with such males also declined by
75% (
Effect of the oral administration of MTZ on the level of serum testosterone (values are mean ± SE of five animals)
Groups | Level of serum testosterone (ng/ml) |
---|---|
2.44 ± 0.2 | |
2.42 ± 0.39 | |
2.14 ± 0.4 | |
2.32 ± 0.25 | |
*; Administration of MTZ for 28 days followed by sacrificing the animals 42 days after cessa- tion of the treatment.
Effect of the oral administration of MTZ on the mating ability and fertility of the males and the females (values are mean ± SE of five males and twelve females)
Groups | Males | Females | ||||
---|---|---|---|---|---|---|
Tested | Mated | Fertile | Tested | Mated | Pregnant | |
6 | 6 | 6 | 12 | 12 | 12 | |
6 | 5 | 2 | 12 | 8 | 3 | |
6 | 6 | 6 | 12 | 8 | 7 | |
*; Administration of MTZ for 28 days followed by sacrificing the animals 42 days after cessation of the treatment.
Effect of the oral administration of MTZ on the number of live blastocysts and pre- and post-implantation loss (values are mean ± SE of twelve females)
Groups | Number of live blastocysts | Pre-implantation loss | Post-implantation loss |
---|---|---|---|
7.25 | 4.25 | 0.41 | |
2.75a | 6.25 | 1.25 | |
4.5 | 5.16 | 0.16 | |
*; Administration of MTZ for 28 days followed by sacrificing the animals 42 days after cessation of the treatment and a; As compared to Group II: p<0.05.
Oral administration of MTZ at therapeutic
and high doses did not affect body weight of
all the animals. However, significant reduction
was noticed in the weight of the testis in the
mice treated with high dose of MTZ. This is
consistent with the findings reported in rats and
mice (
The histological study revealed that the therapeutic dose (250 mg/kgBW) of the drug did not
cause marked alterations in the seminiferous
tubules when administered for 28 days while
the drug at high dose (500 mg/kg BW) for the
same duration induced noticeable regressive
changes in the seminiferous tubules resulting in
the suppression of spermatogenic activity. McClain et al. have reported severe degeneration
of the seminiferous tubules with appearance of
giant cells in their lumina in the testis of rat exposed with MTZ at the dose of 400 mg/kgBW
for 8 weeks. These authors have also reported
partial recovery in spermatogenic activity three
and a half months after cessation of the MTZ
treatment (
The spermatogenic inhibition as noticed in
our study is reflected by alterations in the frequency of different stages as well as diminution of the germ cells at stage VII of the spermatogenic cycle. Earlier studies have shown
that intraperitonial administration of 130 mg/
kgBW of MTZ for 7 days in CFW mice caused
no alterations in the number of stages in the
seminiferous tubules while the number of
cells in stages I, V and XII was significantly
increased (
A significant reduction in the weight of the
epididymis in high dose of MTZ-treated mice
is consistent with that reported by others (
MTZ is reported to inhibit the sperm motility
at different doses in mice and rats (
Significant reduction in the sperm count noticed after high dose of MTZ treatment is consistent with the earlier findings (
In contrast to the reports of El-Nahas and ElAshmawy (
MTZ administration at any dose did not affect the mating ability of the mice. However,
marked reduction was noticed in the fertility of
the males administered only with high dose of
MTZ resulting in decrease in the fertility of females impregnated with such males. A consistent finding is reported in the rat (
High dose of MTZ induced rrelatively reversible deleterious effects on male reproduction and fertility, attributable to the direct action of MTZ on the spermatogenic activity rather than through serum testosterone depletion.