Investigating the Effects of Onion Juice on Male Fertility Factors and Pregnancy Rate After Testicular Torsion/ Detorsion by Intrauterine Insemination Method

Abstract

Introduction Infertility is one of the main problems for the couples. According to statistical prevalence, approximately 15% of the couples are encountered with infertility at the end of the first year of marriage and about 20% of these cases are occurred in males (1). Furthermore, in recent decades, the number of couples searching for consultation and infertility treatment has increased (2). Although most of the men with infertility have an unusual semen analysis and abnormal sperm parameters, the reasons for infertility are not well described (3). However, according to previously research, these reasons may consist of several parameters such as environmental, dietary, medical, genetic as well as physiological factors (4). Meanwhile, as was first described by MacLeod, reactive oxygen species (ROS) has been considered as one of the most common potential causes of infertility in men (5). Similarly, it was shown that high level of ROS was associated with infertility in up to 40% of men (4). Besides, other studies in this regard have mentioned that infertility in 30% to 80% of men was related to the high level of ROS (4,6,7). Oxidative stress (OS) was found to damage the reproductive system and sperm, and therefore result in reducing sperm motility, lipid peroxidation, and oocyte-sperm fusion in addition to increasing the DNA damage (4). ROS, as stated by several studies, had considerable effects on spermatogenesis and sperm function. They reported that overproduction of ROS had harmful effects on motility, morphology, and concentration of sperm and that it caused sperm DNA damage and apoptosis (4,7-9). Testicular torsion, which occurs due to rotation of testicles, is a pathologic condition that leads to acute scrotal pain. According to the reports, although testicular torsion can be observed in any age group, its peak incidence usually occurs in young males aged 13-16 years. Moreover, it is estimated that 4.5 per 100 000 males within the age range of 1-25 years suffer from spermatic cord torsion (10). Furthermore, as pointed out by some studies, the degree and duration of Abstract Objectives: In this experimental study, a testicular torsion/detorsion model was used in male rats to investigate the effect of onion juice on male fertility factors and rate of pregnancy by intrauterine insemination (IUI). Materials and Methods: A total of 56 Wistar rats (28 males vs. 28 females) were included in this study. Male rats were randomly divided into 4 groups including sham, testicular torsion/detorsion, testicular torsion/detorsion treated with onion juice (40 mg/kg, orally) 30 minutes before detorsion, and those rats that were only treated with onion juice. Treatment with onion juice was continued for 14 days after detorsion. At the end of the study, following anesthesia with ketamine/xylazine (5/1 mg/kg), levels of testosterone hormone as well as some oxidative stress markers in the blood serum of the male rats were analyzed. Furthermore, sperm parameters were assayed, and then fertility power of the male rats was investigated in the adult female rats using IUI method. Results: The results of the study showed that there was a significantly negative change in TD group in terms of histological parameters while they were decreased in treated groups. On the one hand, although serum level of malondialdehyde (MDA) significantly increased in TD group, other oxidative markers' (i.e., glutathione peroxidase and superoxide dismutase) serum levels and also testosterone hormone significantly decreased, on the other as compared to the other groups. In addition, the rate of sperm parameters and pregnancy presence were lower in TD group than those of other groups. However, onion juice as a treatment factor could improve the sperm quality and fertility power. Conclusions: The obtained results revealed that using onion juice could enhance the quality of sperm and fertility power after testicular torsion/detorsion. Introduction Throughout the history of the world, the ones who had confronted the bitterest face of poverty and war had always been the women. As known poverty and war affects human health either directly or indirectly, the effects of this condition on health and status of women in the society should not be ignored. This study intends to cast light on the effects of war and poverty on the reproductive health of women. For this purpose, the face of war affecting the women, the problem of immigration, inequalities in distribution of income based on gender and the effects of all these on the reproductive health of women will be addressed.

Full text

Introduction
Infertility is one of the main problems for the couples.
According to statistical prevalence, approximately 15%
of the couples are encountered with infertility at the end
of the first year of marriage and about 20% of these cases
are occurred in males (1). Furthermore, in recent decades,
the number of couples searching for consultation and
infertility treatment has increased (2). Although most of
the men with infertility have an unusual semen analysis and
abnormal sperm parameters, the reasons for infertility are
not well described (3). However, according to previously
research, these reasons may consist of several parameters
such as environmental, dietary, medical, genetic as well
as physiological factors (4). Meanwhile, as was first
described by MacLeod, reactive oxygen species (ROS) has
been considered as one of the most common potential
causes of infertility in men (5). Similarly, it was shown
that high level of ROS was associated with infertility in
up to 40% of men (4). Besides, other studies in this regard
have mentioned that infertility in 30% to 80% of men was
related to the high level of ROS (4,6,7). Oxidative stress
(OS) was found to damage the reproductive system and
sperm, and therefore result in reducing sperm motility,
lipid peroxidation, and oocyte-sperm fusion in addition to
increasing the DNA damage (4). ROS, as stated by several
studies, had considerable effects on spermatogenesis
and sperm function. They reported that overproduction
of ROS had harmful effects on motility, morphology,
and concentration of sperm and that it caused sperm
DNA damage and apoptosis (4,7-9). Testicular torsion,
which occurs due to rotation of testicles, is a pathologic
condition that leads to acute scrotal pain. According to
the reports, although testicular torsion can be observed in
any age group, its peak incidence usually occurs in young
males aged 13-16 years. Moreover, it is estimated that
4.5 per 100 000 males within the age range of 1-25 years
suffer from spermatic cord torsion (10). Furthermore, as
pointed out by some studies, the degree and duration of
Abstract
Objectives: In this experimental study, a testicular torsion/detorsion model was used in male rats to investigate the effect of onion
juice on male fertility factors and rate of pregnancy by intrauterine insemination (IUI).
Materials and Methods: A total of 56 Wistar rats (28 males vs. 28 females) were included in this study. Male rats were randomly
divided into 4 groups including sham, testicular torsion/detorsion, testicular torsion/detorsion treated with onion juice (40 mg/kg,
orally) 30 minutes before detorsion, and those rats that were only treated with onion juice. Treatment with onion juice was continued
for 14 days after detorsion. At the end of the study, following anesthesia with ketamine/xylazine (5/1 mg/kg), levels of testosterone
hormone as well as some oxidative stress markers in the blood serum of the male rats were analyzed. Furthermore, sperm parameters
were assayed, and then fertility power of the male rats was investigated in the adult female rats using IUI method.
Results: The results of the study showed that there was a significantly negative change in TD group in terms of histological
parameters while they were decreased in treated groups. On the one hand, although serum level of malondialdehyde (MDA)
significantly increased in TD group, other oxidative markers’ (i.e., glutathione peroxidase and superoxide dismutase) serum levels
and also testosterone hormone significantly decreased, on the other as compared to the other groups. In addition, the rate of sperm
parameters and pregnancy presence were lower in TD group than those of other groups. However, onion juice as a treatment factor
could improve the sperm quality and fertility power.
Conclusions: The obtained results revealed that using onion juice could enhance the quality of sperm and fertility power after
testicular torsion/detorsion.
Keywords: Torsion/detorsion, Onion, Allium cepa, Testis, Fertility, IUI
Investigating the Effects of Onion Juice on Male Fertility
Factors and Pregnancy Rate After Testicular Torsion/
Detorsion by Intrauterine Insemination Method
Majid Shokoohi1
ID
, Elahe Olad Saheb Madarek1, Arash Khaki1*
ID
, Hamed Shoorei1, Amir Afshin Khaki1,
Malihe Soltani2, Nava Ainehchi1
Open Access Original Article
International Journal of Women’s Health and Reproduction Sciences
Vol. 6, No. 4, October 2018, 499–505
http://www.ijwhr.net doi 10.15296/ijwhr.2018.82
ISSN 2330- 4456
Received 2 April 2018, Accepted 11 September 2018, Available online 3 October 2018
1Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences (TUOMS), Tabriz, Iran. 2Department of Basic Sciences,
Faculty of Medicine, Gonabad University of Medical Sciences, Gonabad, Iran.
*Corresponding Author: Arash Khaki, Tel: +989143138399, Email: arashkhaki@yahoo.com
Introduction
Throughout the history of the world, the ones who had
confronted the bitterest face of poverty and war had al-
ways been the women. As known poverty and war affects
human health either directly or indirectly, the effects of
this condition on health and status of women in the so-
ciety should not be ignored. This study intends to cast
light on the effects of war and poverty on the reproductive
health of women. For this purpose, the face of war affect-
ing the women, the problem of immigration, inequalities
in distribution of income based on gender and the effects
of all these on the reproductive health of women will be
addressed.
War and Women’s Health
Famine, synonymous with war and poverty, is clearer for
women; war means deep disadvantages such as full de-
struction, loss of future and uncertainty for women. Wars
are conflicts that destroy families, societies and cultures
that negatively affect the health of community and cause
violation of human rights. According to the data of World
Health Organization (WHO) and World Bank, in 2002
wars had been among the first ten reasons which killed
the most and caused disabilities. Civil losses are at the rate
of 90% within all losses (1).
War has many negative effects on human health. One of
these is its effect of shortening the average human life.
According to the data of WHO, the average human life is
68.1 years for males and 72.7 years for females. It is being
thought that severe military conflicts in Africa shorten
the expected lifetime for more than 2 years. In general,
WHO had calculated that 269 thousand people had died
in 1999 due to the effect of wars and that loss of 8.44 mil-
lion healthy years of life had occurred (2,3).
Wars negatively affect the provision of health services.
Health institutions such as hospitals, laboratories and
health centers are direct targets of war. Moreover, the wars
cause the migration of qualified health employees, and
thus the health services hitches. Assessments made indi-
cate that the effect of destruction in the infrastructure of
health continues for 5-10 years even after the finalization
of conflicts (3). Due to resource requirements in the re-
structuring investments after war, the share allocated to
health has decreased (1).
Mortalities and Morbidities
The ones who are most affected from wars are women and
children. While deaths depending on direct violence af-
fect the male population, the indirect deaths kill children,
women and elders more. In Iraq between 1990-1994, in-
fant deaths had shown this reality in its more bare form
with an increase of 600% (4). The war taking five years
increases the child deaths under age of 5 by 13%. Also 47%
of all the refugees in the world and 50% of asylum seekers
and displaced people are women and girls and 44% ref-
ugees and asylum seekers are children under the age of
18 (5).
As the result of wars and armed conflicts, women are
Abstract
War and poverty are ‘extraordinary conditions created by human intervention’ and ‘preventable public health problems.’ War and
poverty have many negative effects on human health, especially women’s health. Health problems arising due to war and poverty are
being observed as sexual abuse and rape, all kinds of violence and subsequent gynecologic and obstetrics problems with physiological
and psychological courses, and pregnancies as the result of undesired but forced or obliged marriages and even rapes. Certainly,
unjust treatment such as being unable to gain footing on the land it is lived (asylum seeker, refugee, etc.) and being deprived of
social security, citizenship rights and human rights brings about the deprivation of access to health services and of provision of
service intended for gynecology and obstetrics. The purpose of this article is to address effects of war and poverty on the health of
reproduction of women and to offer scientific contribution and solutions.
Keywords: Poverty, Reproductive health, War
Women on the Other Side of War and Poverty: Its Effect
on the Health of Reproduction
Ayse Cevirme1, Yasemin Hamlaci2*, Kevser Ozdemir2
Open Access Review
International Journal of Women’s Health and Reproduction Sciences
Vol. 3, No. 3, July 2015, 126–131
Received 12 December 2014, Accepted 25 April 2015, Available online 1 July 2015
1Department of Nursing, Sakarya University, Sakarya, Turkey. 2Department of Midwifery, Sakarya University, Sakarya, Turkey.
*Corresponding author: Yasemin Hamlaci, Department of Midwifery, Sakarya University, Sakarya, Turkey. Tel: +905556080628,
Email: yaseminhamlaci@gmail.com
http://www.ijwhr.net doi 10.15296/ijwhr.2015.27
ISSN 2330- 4456

Shokoohi et al
International Journal of Women’s Health and Reproduction Sciences, Vol. 6, No. 4, October 2018
500
torsion have been considered as two important predictors
of testicular damage (11,12), If detorsion was performed
within 6 hours of torsion, testicles could be saved in 90%
of the cases; however, if the time of detorsion procedure
increased to 12-24 hours, the maintenance rate of
testicles decreased to 50% and 10%, respectively (13).
It was also reported that the twisted spermatic cord led
to a decrease in/or even complete loss of the perfusion
flow to the affected testis and other scrotal contents.
Therefore, an emergency surgery for reperfusion of the
affected testis was needed. In addition, any attempt to
reperfusion of ischemic tissue was considered as the cause
of further damage for the testis. For example, damage in
the epithelium of seminiferous tubules and loss of germ
cells after ischemia/reperfusion (IR) injury of the testis
had been reported in some studies (10,14). Moreover, a
possible cause of damage in testicles may be a produced
ROS during the process of ischemia/reperfusion (15).
Nowadays, using a different range of antioxidants has been
regarded as a new method in treatment of male infertility.
Allium cepa (onion) is one of the natural antioxidants that
has been used in traditional medicine for thousands of
years. The extract of onion protects sperm against DNA
damage and other important molecules related to OS. It
also improves sperm quality and enhances the power of
fertility (16). Hence, the present study aimed to evaluate
the effect of onion juice on rate of male fertility and sperm
quality after testicular torsion/detorsion by intrauterine
insemination (IUI) in adult female rats.
Materials and Methods
The present experimental study was carried out on 56
Wistar rats (including 28 adult males vs. 28 adult females)
weighed between 250-300 g. Rats were maintained in the
animal house of Tabriz University of Medical Sciences
under the standard conditions namely, the temperature
23±2°C, humidity 60%-70%, and a 12/12-hour light/dark
cycle while having free access to enough food and water.
Male rats were classified into 4 groups (n = 7) including
sham (G1), testicular torsion for 4 hours followed by
surgical detorsion (TD/G2), TD received onion juice (40
mg/kg, orally) 30 minutes prior to detorsion (TDOJ/G3),
and healthy rats that only received (40 mg/kg) onion juice
(OJ/G4).
Surgical Procedure
In the surgical process, male rats were first anesthetized
using ketamine/xylazine (5/1 mg/kg), then left testis
was exposed and rotated 720° counterclockwise by an
incision on the scrotum. Afterwards, the tunica albuginea
of the torsioned testicle was sutured to the dartos muscle
using three 6/0 silk. In addition, the scrotum was closed
employing 5/0 silk sutures. The duration of torsion was
4 hours, thereafter another surgery was performed for
detorsion. Fourteen days post procedure, all male rats
were anesthetized and their blood was drawn from
the superior vena cava (IVC) vein, then, the obtained
blood was centrifuged at 3000 rpm (for 10 minutes) and
afterwards the serum was separated and kept at –70°C to
analyze the levels of testosterone hormone and some stress
oxidative markers. Moreover, on the last day of the study,
that is day 14, male rats underwent bilateral orchiectomy
and their left testicles were fixed in Bouin’s fluid (17, 18).
The Procedure of IUI
A) Sperm Extraction From Epididymis: after reperfusion
period, all male rats were anesthetized, then the tail of
epididymis was quickly removed and placed into the
5.0 mL of phosphate buffer saline (PBS) supplemented
with 10 mg/mL bovine albumin. Several cuttings were
made with scissors in the epididymis and the sperm was
allowed to get out and float for 5 minutes. Then, some of
the suspension was removed to examine the morphology,
motility, and count of the sperm. The suspension of sperm
was kept in the incubator (37°C) for 20 minutes until
insemination (19).
B) Insemination: female rats were first anesthetized, then
the caudal part of the uterus was exposed by an incision
in the low midline of the abdomen. The sperm suspension
(0.1 mL) was injected into each horn of uterine lumen and
incision was closed with 5/0 silk sutures (19).
Counting of Embryos
Three days following insemination, female rats were
anesthetized with ketamine and xylazine. Fallopian tubes,
the rostral portion of uterine horns, and both ovaries were
removed and placed in a plate. Then, a 27-gauge needle
was put into the infundibulum of the uterine horn and
afterwards, the preimplantation embryos were gathered
by PBS flushing and their number was counted under
a stereomicroscope. Female rats were considered not
pregnant when there were unfertilized oocytes while in
the pregnant rats there was at least one embryo (19).
Tissue Preparation
Removed testicles were placed into falcon tubes,
containing Bouin’s solution, for 48 hours. Then, the steps
of tissue passage were carried out and paraffin embedded
testicles were cut and thus, slides with 5 µM thickness
were prepared. Each slide was stained with hematoxylin
and eosin (H & E), then examined under an optical
microscope (Nikon, Japan) at the magnification of 400X
(20,21).
Histopathological Evaluations
To evaluate the histological changes in seminiferous
tubules of testicles after fixation, the testicles were
dehydrated with an ascending ethanol sequence, cleared
with xylene and embedded in paraffin. Spermatogenesis
in the seminiferous tubules was evaluated with Johnson’s
score. To this end, 50 seminiferous tubules were randomly
selected from each slide and Johnson’s score (scales of

Shokoohi et al
International Journal of Women’s Health and Reproduction Sciences, Vol. 6, No. 4, October 2018 501
1–10 based on the level of spermatogenesis) was calculated
for each tubule. Then, the mean Johnson’s score of each
case was computed (20). Morphometric studies were
conducted on the seminiferous tubules. For this purpose,
in each slide, 30 round or nearly round seminiferous
tubules were randomly selected, then their morphometry
was investigated under the microscope using the linear
eyepiece grids at the magnification of 400X. The internal
diameter of the seminiferous tubules was measured by
the mean average of two perpendicular diameters from
the basement membrane on one side of the tubule to the
other side of the tubule. The height (thickness) of the
germinal epithelium (from base membrane to lumen) was
calculated and the data was presented as micrometer (µM)
(20).
Measurement of Malondialdehyde, Superoxide Dismutase,
and Glutathione Peroxidase Serum Levels
The serum levels of Malondialdehyde (MDA), Superoxide
Dismutase (SOD), and Glutathione Peroxidase (GPx)
were measured based on the previous study conducted
by the researchers of the present study (21). Briefly, 200
lambda (λ) of serum was poured into a tube containing
3000 λ of glacial acetic acid, 3000 λ of NaOH (2%), and
tetrabutylamine (TBA, 1%). Then, the tube was placed
into a boiling water bath for 15 minutes. After cooling, the
absorbance of the product was read at 532 nm. Moreover,
the serum activities of SOD and GPx were measured
according to the protocol of the kits (Randox, UK).
Serum Testosterone Measurement
Serum levels of testosterone were measured according to the
protocol of testosterone enzyme-linked immunosorbent
assay (ELISA) kit (Demeditec Diagnostices, Germany).
The absorbance of the product was read at 405 nm.
Sperm Counting
The left epididymis was minced in 5 mL PBS (pH = 7.2).
Then, a new solution was made by dissolving 100 λ of
sperm solution in 900 λ of PBS. At the next step, 1 drop of
the solution was poured into the Neubauer chamber and
the number of sperm was counted based on the World
Health Organization (WHO) protocol (9, 22).
Morphology of Sperm
After preparing the smears of sperm for accessing to the
morphology, the slides were dried exposing to the air,
fixed with alcohol 96%, and stained with Hand E. Then,
in each slide, 150 sperms were randomly counted and
therefore, based on the WHO protocol, the percentage of
the normal and abnormal sperms were determined (9,22).
Statistical Analysis
To analyze the data, SPSS (statistical package for the social
sciences) software, version 18 (IBM, USA) was used.
The normality of data was determined by Kolmogorov-
Smirnov test. In order to compare the data, one-way
ANOVA was carried out followed by post-hoc Tukey
HSD test and all the data were presented as mean ±
standard error (SE). The level of P < 0.05 was considered
as statistically significant.
Results
Histopathological Parameters of Testis
Table 1 and Figure 1 show histopathological parameters of
the testis. The mean Johnson’s score (MJS), the diameter
of seminiferous tubules (STD), and the thickness of
seminiferous tubule epithelium (HE) significantly
decreased in the torsion/detorsion group as compared to
the sham group. These parameters increased in treated
groups that received onion juice (P < 0.05).
Testosterone
The serum level of testosterone was significantly higher in
TD group than that of the sham group (P < 0.05). However,
in the treated groups, it was significantly higher than TD
group (Figure 2).
Oxidative Stress Markers
In TD group, serum levels of GPX and SOD were
significantly declined in comparison with the sham group
(P < 0.05). In the treated groups, that is, TDOJ and OJ,
serum levels of SOD and GPX increased significantly
(P < 0.05). In addition, although the serum level of MDA
was significantly higher in G2 than G1, it decreased
(P≤0.01) in the treated groups (Table 2).
Table 1. Comparison of Testicular Mean Johnson’s Score, Seminiferous Tubule Diameter, and Height of Epithelium in All Groups of the Study
Groups Mean Johnsen’s Score STD HE
Sham 9.67 ± .065 267.42 ± 1.41 72.12 ± 3.21
TD 4.50 ±.075*140.80 ± 1.63*29.82 ± 4.32*
TDOJ 7.12 ± 0.045a208.75 ± 7.13a57.45 ± 3.45a
OJ 9.85 ± 0.075a273.52 ± 4.30a75.16 ± 2.31a
Note 1. Sham, control group without the applicaon of tors1ion; TD, tescular torsion/detorsion group; TDOJ, tescular torsion/detorsion, then
received 40 mg/kg of onion juice 30 minutes before detorsion; OJ, healthy rats received 40 mg/kg of onion juice; STD, seminiferous tubule diameter;
HE, height of epithelium.
Note 2. All the data are displayed as Mean ± SE; the asterisk (*) and symbol (a) show signicant dierence with the sham and TD groups,respecvely.
*(P<0.05).

Shokoohi et al
International Journal of Women’s Health and Reproduction Sciences, Vol. 6, No. 4, October 2018
502
Parameters of Sperm
Semen analysis in all of the studied groups revealed that
there was a significant decrease in the number of sperm
in G2 as compared with G1 while in therapeutic groups
as compared to G2, it increased significantly (P < 0.02).
Furthermore, evaluating the morphology of the sperm in
Figure 1: Histological Findings in All Groups of the Study.
Note 1. A: Sham, control group without the application of torsion; B:
TD, testicular torsion/detorsion group; C: TDOJ, testicular torsion/
detorsion, then received 40 mg/kg of onion juice 30 minutes before
detorsion; D: OJ, healthy rats received 40 mg/kg of onion juice. Note
2. Scale bar: X100 (100 μM)
Figure 3. Morphology of the Sperm. Note 1. Symbols a and b show
normal and abnormal sperms, respectively; Note 2. Scale Bar: X100
(100 μM).
Figure 2: Comparison of Testosterone Levels in All Studied Groups.
Note 1. Sham, control group without the application of torsion; TD,
testicular torsion/detorsion group; TDOJ, testicular torsion/detorsion,
then received 40 mg/kg of onion juice 30 minutes before detorsion;
OJ, healthy rats received 40 mg/kg of onion juice
Note 2. The asterisk (*) and symbol (a) demonstrate significant
difference with the sham and TD groups, respectively. *(P<0.05)
100 μM
*
*a
a
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Sham TD TDOJ OJ
Serum Testosteron Levels
Group
Table 2. Comparison of Sperm Parameters in All the Studied Groups
Groups Concentraon
(Mean ± SEM)
Normal Morphology
(Mean ± SEM)
Normal Molity
(Mean ± SEM)
Sham 30.45 ± 2.11 65.65% ± 5.08 63.11% ± 2.31
TD 13.25 ± 3.24*18.35% ± 2.35*17.62% ± 3.49*
TDOJ 23.55 ± 2.17a54.34% ± 4.21a52.71% ± 2.21a
OJ 36.36 ± 1.08a68.46% ± 2.08a65.34% ± 3.14a
Note 1. Sham, control group without the application of torsion; TD, testicular torsion/detorsion group;TDOJ, testicular torsion/detorsion, then received
40 mg/kg of onion juice 30 minutes before detorsion; OJ, healthy rats received 40 mg/kg of onion juice.
Note 2. The asterisk (*) and symbol (a) represent significant difference with the sham group and TD groups, respectively. *(P<0.05)
different groups indicated that the percentage of abnormal
sperm was higher in torsion/detorsion group than that of
the sham group. However, in TDOJ and OJ groups, the
percentage of abnormal sperm decreased compared to the
torsion/detorsion group (P ≤ 0.001). Besides, assessment
of sperm motility showed that it was significantly lower
in G2 than G1, but it increased (P ≤ 0.001) in the treated
groups (Table 3, Figure 3).
The Rate of Pregnancy and the Number of Embryos
The pregnancy rate through insemination of sperm in the
sham and testicular torsion/detorsion groups revealed that
all rats in the sham group were pregnant while in TD group,
only one rat, among others, was pregnant; therefore, a
significant decrease was observed (P < 0.02) in this regard.
Moreover, after counting the number of embryos in TD
group, a significant difference was observed compared to
the sham group (P < 0.01). Furthermore, in TDOJ group, 5
rats out of all (n = 7) were pregnant while in OJ group, all
the rats (n = 7) were pregnant; hence, the rate of pregnancy
in TDOJ and OJ groups was significantly higher (P < 0.02)
than TD group (Table 4, Figure 4).
Discussion
Testicular torsion is one of the main causes of urologic
emergency. At the moment, in order to preserve this
condition and also preservation of the testis and fertility,
quick diagnosis as well as surgical procedure to detorsion
are needed (23). The injury of testicles induced by IR
100 μM

Shokoohi et al
International Journal of Women’s Health and Reproduction Sciences, Vol. 6, No. 4, October 2018 503
had been considered as the main pathophysiology of
testicular torsion (23). The IR damage can induce a
pathophysiologic cascades containing inflammatory
responses with extrication of cytokines as well as ROS
production (24). One research showed that severity of
ischemic histological damage had a direct relationship
with the duration and degree of torsion (25). Previous
researches on testicular torsion in adult rats demonstrated
that duration of 4 hours, 720° rotation of the spermatic
cord, and detorsion caused a significant overproduction
in the levels of nitric oxide content, lipid peroxidation,
and myeloperoxidase activity (an indicator of neutrophil
accumulation) in the testicles (20,21,26). This resulted in
the permanent loss of spermatogenesis (27). It has been
reported that ischemia followed by reperfusion can induce
tissue damages by several mechanisms including increased
level of ROS and diffusion of inflammatory factors
such as diffusion of apoptotic caspase cascade enzymes
Table 3. Comparison of Intrauterine Insemination Results in All the Studied Groups
Groups Pregnancy Rate Number of Embryo in Le Horne Number of Embryo in Right Horne
Sham 100% 4 ± 0.81 4 ± 0.86
TD 14.28% 0.14 ± 0.37*1 ± 0.89*
TDOJ 71.42%a1.85 ± 1.46a1.85 ± 1.34a
OJ 100%a4.42 ± 1.27a4.71 ± 0.75a
Note 1. Sham, control group without the applicaon of torsion; TD, tescular torsion/detorsion group; TDOJ, tescular torsion/detorsion, then received
40 mg/kg of onion juice 30 minutes before detorsion; OJ, healthy rats received 40 mg/kg of onion juice.
Note 2. The asterisk (*) and symbol (a) denote signicant dierence with the sham group and TD groups, respecvely. *(P<0.05)
Table 4. Blood Levels of SOD (U/mL), GPX (U/mL), MDA (nM) in Different Groups of the Study
Groups SOD
(Mean ± SEM)
GPx
(Mean ± SEM)
MDA
(Mean ± SEM)
Sham 2.07 ± 0.11 22.74 ± 5.11 1. 78 ± 0.11
TD 0.97 ± 0.09*9.34 ± 7.32*2.72 ± 0.29*
TDOJ 1.75 ± 0.17a18.24 ± 4.21a1.43 ± 021a
OJ 2.11 ± 0.08a23.76 ± 6.08a1.42 ± 0.14a
Note 1. Sham, control group without the applicaon of torsion; TD, tescular torsion/detorsion group; TDOJ, tescular torsion/detorsion, then received
40 mg/kg of onion juice 30 minutes before detorsion; OJ, healthy rats received 40 mg/kg of onion juice.
Note 2. The asterisk (*) and symbol (a) show signicant dierence with the sham group and the TD groups, respecvely. *(P<0.05)
Figure 4: IUI, Intrauterine Insemination in Adult Female Rats
Figure 4. IUI, Intrauterine Insemination in Adult Female Rats.
by decreasing the blood flow (26). Similarly, Yuluğ et
al reported that ischemia (for 4 hours) and afterward
reperfusion injury (for 24 hours) could cause testicular
tissue damage (26). Therefore, according to previous
studies, the ischemia period was selected 4 hours with 14
days reperfusion. Furthermore, findings of the present
study showed that 720 degree ischemia (for 4 hours) and
afterward reperfusion (for 14 days) led to severe edema
and density of blood vessels. In addition, histopathological
examinations revealed that testicular torsion/detorsion
resulted in degeneration of germ cells layers and decrease
of Johnson’s score, the diameter of seminiferous tubules,
and the thickness of the epithelium of seminiferous
tubules. According to the degenerative damages in
testicular tissue induced by torsion/detorsion, it was
observed that serum levels of testosterone significantly
decreased in testicular torsion/detorsion group. This
phenomenon was probably related to the overproduction
of ROS, apoptosis of germ cells, and/or injury to the Leydig
cells of the testis (26,27). In addition, in the previous study
by the authors of the present research, testicular torsion
(for 5 hours) followed by detorsion could decrease the
serum levels of testosterone (20). In the present study, it
was also observed that testicular torsion/detorsion caused
a decrease in the quality of sperm including motility and
the count of sperm. Moreover, the number of abnormal
sperm increased in TD group; this was maybe related to
the decrease of testosterone level, apoptosis in testicular
germ cells and also ROS production. The results of the
current study are in agreement with the findings of the
study by Visser et al which showed that testicular torsion

Shokoohi et al
International Journal of Women’s Health and Reproduction Sciences, Vol. 6, No. 4, October 2018
504
(for 4 hours) and subsequent reperfusion decreased
the quality of sperm (28). Furthermore, Kurcer et al in
their study found that testicular ischemia/reperfusion
decreased the quality of sperm including a reduction in
sperm concentration and an increase in the number of
sperm with abnormal morphology and motility (29).
Besides, in the present study, following testicular torsion/
detorsion, fertility power by IUI, the rate of pregnancy,
and also the number of embryos were measured. However,
the pregnancy rate and the number of embryos were
significantly decreased in testicular torsion/detorsion
group. This probably occurred due to the reduction of
serum testosterone level and sperm quality. Additionally,
several studies have shown that ROS had deleterious effects
on spermatogenesis and sperm function. In other words,
the overproduction of ROS could lead to the reduction of
sperm concentration and sperm quality (4,7,8).
In the present research, measuring the generalized
OS markers (i.e., MDA, SOD, and GPx), the results
showed that testicular torsion/detorsion increased the
lipid peroxidation (serum level of MDA) and decreased
the activities of superoxide dismutase and glutathione
peroxidase. Several studies have confirmed that testicular
torsion/detorsion increased the OS marker and reduced
the levels of antioxidant enzymes (24,26,27).In addition,
the results of the previous study by the present researchers
showed that testicular torsion (for 5 hours) followed by
detorsion increased MDA level and decreased the SOD
and GPx activities (20).
The findings of this study demonstrated that onion juice
could protect the testis tissue against damages induced
by testicular torsion/detorsion. In this regard, the mean
Johnson’s score, the diameter of seminiferous tubules, and
the thickness of the epithelium of seminiferous tubules
in treated groups that received onion juice significantly
increased as compared to the testicular torsion/detorsion
group. It is suggested that antioxidant properties of onion
juice and its compounds such as phenolic compositions
and flavonoids can prevent overproduction of ROS (30).
In the same vein, some studies reported that extract of
onion could protect the testis tissue against oxidative
damages induced by permethrin (16,30). Besides, Ige
et al in their study showed that Allium cepa (AC) could
protect the testis tissue against cadmium toxicity (31). The
results of this study also demonstrated that onion juice
had an improving effect on serum levels of testosterone.
The quality of sperm such as the motility, count, and the
percentage of normal morphological sperm increased
as well. These phenomena may have occurred due to
protective effects of onion juice against the damage of the
Leydig cells (30). Similarly, one study indicated that onion
juice had a positive impacts on the sexual hormones in rats
treated with the antiepileptic drug. It also could increase
the fertility power (32). In the current study, evaluating
the fertility power, it was found that pregnancy rate in
TDOJ and OJ groups increased in comparison with the
testicular torsion/detorsion group. In agreement with the
results of the present study, khaki et al also reported that
the use of onion juice could increase the concentration
and motility of sperm and decrease the percentage of
sperm with abnormal morphology (16,30,32).
The results of the present study showed that onion juice
resulted in a decline of lipid peroxidation (serum level of
MDA) and an increase in (SOD and GPx activities. Several
studies have confirmed that AC extract can reduce the OS
and increase the level of the antioxidant enzyme (33,34).
Conclusions
According to the findings, it was found that testicular
torsion/detorsion, as a urological and emergencial
condition, caused testis tissue damage and increased
lipid peroxidation level. Moreover, this situation led to a
decrease in serum levels of testosterone, sperm quality,
and male fertility power. Therefore, it can be concluded
that male infertility was one of the most important side
effects of testicular torsion followed by detorsion. In
addition, consumption of onion juice could protect the
testis tissue against damages induced by testicular torsion/
detorsion and increase fertility power of men.
Conflict of Interests
At the present time, AK acts as the Editor of the journal.
As a result of this statement, it is declared that his relation
with the journal has affected neither the peer-review
process nor the acceptance of the manuscript. The rest
declare no conflict of interest.
Ethical Issues
Tabriz University of Medical Sciences approved the
study (with the ethical code of IR.TBZMED.VCR.
REC.1397.126).
Financial Support
This study was financially supported by Women’s
Reproductive Health Research Center of Tabriz University
of Medical Sciences.
Acknowledgments
The authors would appreciate the stuff of Women’s
Reproductive Health Research Center of Tabriz University
of Medical Sciences.
References
1. Moradi M, Moradi A, Alemi M, et al. Safety and ecacy
of clomiphene citrate and L-carnitine in idiopathic male
infertility: a comparative study. Urol J. 2010;7(3):188-193.
2. Rabin DS, Qadeer U, Steir VE. A cost and outcome model
of fertility treatment in a managed care environment. Fertil
Steril 1996;66(6):896-903.
3. Agarwal A, Sekhon LH. e role of antioxidant therapy
in the treatment of male infertility. Hum Fertil (Camb).

Shokoohi et al
International Journal of Women’s Health and Reproduction Sciences, Vol. 6, No. 4, October 2018 505
2010;13(4):217-225. doi:10.3109/14647273.2010.532279
4. Ko EY, Sabanegh ES, Jr., Agarwal A. Male infertility testing:
reactive oxygen species and antioxidant capacity. Fertil Steril.
2014;102(6):1518-1527. doi:10.1016/j.fertnstert.2014.10.020
5. MacLeod J. e role of oxygen in the metabolism and
motility of human spermatozoa. American Journal of
Physiology--Legacy Content. 1943;138(3):512-518.
6. Makker K, Agarwal A, Sharma R. Oxidative stress & male
infertility. Indian J Med Res. 2009;129(4):357-367.
7. Tremellen K. Oxidative stress and male infertility—a clinical
perspective. Hum Reprod Update. 2008;14(3):243-258.
8. Agarwal A, Sharma RK, Sharma R, et al. Characterizing
semen parameters and their association with reactive
oxygen species in infertile men. Reprod Biol Endocrinol.
2014;12(1):33. doi:10.1186/1477-7827-12-33
9. Shokoohi M, Shoorei H, Soltani M, Abtahi‐Eivari SH,
Salimnejad R, Moghimian M. Protective eects of the
hydroalcoholic extract of Fumaria parviora on testicular
injury induced by torsion/detorsion in adult rats.
Andrologia. 2018:e13047. doi: 10.1111/and.13047.
10. Rabani R, Sadeghipour-Roodsari H, Sepehri H,
Dehpour AH-SA. Eect of cannabinoids on testicular
Ischemia-reperfusion injury in rat. Acta Medica Iranica.
2006;44(6):365-370.
11. Barada JH, Weingarten JL, Cromie WJ. Testicular salvage
and age-related delay in the presentation of testicular
torsion. J Urol. 1989;142(3):746-748.
12. Sessions AE, Rabinowitz R, Hulbert WC, Goldstein
MM, Mevorach RA. Testicular torsion: direction, degree,
duration and disinformation. J Urol. 2003;169(2):663-665.
doi:10.1097/01.ju.0000047381.36380.0e
13. Sharp VJ, Kieran K, Arlen AM. Testicular torsion:
diagnosis, evaluation, and management. Am Fam Physician.
2013;88(12):835-840.
14. Lysiak JJ, Turner SD, Nguyen QA, Singbartl K, Ley K,
Turner TT. Essential role of neutrophils in germ cell-specic
apoptosis following ischemia/reperfusion injury of the
mouse testis. Biol Reprod. 2001;65(3):718-725.
15. Taati M, Moghadasi M, Dezfoulian O, Asadian P,
Zendehdel M. Eects of Ghrelin on germ cell apoptosis
and proinammatory cytokines production in Ischemia-
reperfusion of the rat testis. Iran J Reprod Med.
2015;13(2):85-92.
16. Khaki A, Rajabzadeh A, Khaki AA. Side Eects of
Pyrethroid and Supporting Role of Onion in the Male Rat’s
Spermatogenesis. Chinese Med J. 2017;130(24):3015.
17. Ozbal S, Ergur BU, Erbil G, Tekmen I, Bagrıyanık A, Cavdar
Z. e Eects of α-Lipoic Acid against Testicular Ischemia-
Reperfusion Injury in Rats. ScienticWorldJournal.
2012;2012:489248. doi:10.1100/2012/489248
18. Rashed FK, Ghasemi B, Deldade Mogaddam H, Mesgari
M. e eect of erythropoietin on ischemia/reperfusion
injury aer testicular torsion/detorsion: a randomized
experimental study. ISRN Urol. 2013;2013:351309.
doi:10.1155/2013/351309
19. Cukierski MA, Sina JL, Prahalada S, Robertson RT. Eects
of seminal vesicle and coagulating gland ablation on fertility
in rats. Reprod Toxicol. 1991;5(4):347-352.
20. Moghimian M, Soltani M, Abtahi H, Shokoohi M. Eect of
vitamin C on tissue damage and oxidative stress following
tunica vaginalis ap coverage aer testicular torsion. J
Pediatr Surg. 2017;52(10):1651-1655.
21. Moghimian M, Abtahi-Evari S-H, Shokoohi M, Amiri M,
Soltani M. Eect of Syzygium aromaticum (clove) extract
on seminiferous tubules and oxidative stress aer testicular
torsion in adult rats. Physiol Pharmacol. 2017;21(4):343-
350.
22. Benny B, Doss CGP, Kumar DT, Devi SA. Assessing
reproductive toxicity and antioxidant enzymes on beta
asarone induced male Wistar albino rats: In vivo and
computational analysis. Life Sci. 2017;173:150-160.
23. Lee J-W, Kim J-I, Lee Y-A, et al. Inhaled hydrogen gas
therapy for prevention of testicular ischemia/reperfusion
injury in rats. J Pediatr Surg. 2012;47(4):736-742.
24. Ayan M, Tas U, Sogut E, et al. Protective eect of
thymoquinone against testicular torsion induced oxidative
injury. Andrologia. 2016;48(2):143-151.
25. Yang C, Song B, Tan J, Liu X, Wei GH. Testicular torsion in
children: a 20-year retrospective study in a single institution.
ScienticWorldJournal. 2011;11:362-8. doi: 10.1100/
tsw.2011.39.
26. Yuluğ E, Türedi S, Karagüzel E, Kutlu Ö, Menteşe A, Alver A.
e short term eects of resveratrol on ischemia–reperfusion
injury in rat testis. J Pediatr Surg. 2014;49(3):484-489.
27. Ghasemnezhad R, Mohammadghasemi F, Faghani M,
Bahadori MH. Oxytocin can decrease germ cells apoptotic
index in testis under acute ischemia reperfusion in a rat
model. Iran J Reprod Med. 2015;13(5):283.
28. Visser A, Heyns C. Testicular function aer torsion of the
spermatic cord. BJU Int. 2003;92(3):200-203.
29. Kurcer Z, Hekimoglu A, Aral F, Baba F, Sahna E. Eect of
melatonin on epididymal sperm quality aer testicular
ischemia/reperfusion in rats. Fertil Steril. 2010;93(5):1545-
1549.
30. Khaki A, Khaki AA, Rajabzadeh A. e eects of
Permethrin and antioxidant properties of Allium cepa
(onion) on testicles parameters of male rats. Toxin Reviews.
2017;36(1):1-6.
31. Ige SF, Olaleye SB, Akhigbe RE, Akanbi TA, Oyekunle OA,
Udoh UAS. Testicular toxicity and sperm quality following
cadmium exposure in rats: Ameliorative potentials of Allium
cepa. J Hum Reprod Sci. 2012;5(1):37-42. doi:10.4103/0974-
1208.97798
32. Khaki A, Farnam A, Badie AD, Nikniaz H. Treatment eects
of onion (Allium cepa) and Ginger (Zingiber ocinale)
on sexual behavior of rat aer inducing an antiepileptic
drug (lamotrigine). Balkan Med J. 2012;29(3):236-242.
doi:10.5152/balkanmedj.2012.045
33. Achary VMM, Jena S, Panda KK, Panda BB. Aluminium
induced oxidative stress and DNA damage in root cells of
Allium cepa L. Ecotoxicol Environ Saf. 2008;70(2):300-310.
34. Fatima RA, Ahmad M. Certain antioxidant enzymes of
Allium cepa as biomarkers for the detection of toxic heavy
metals in wastewater. Sci Total Environ. 2005;346(1-3):256-
273.
© 2018 The Author (s); This is an open-access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.