Cryopreservation of human semen is considered one of
the most vital and essential strategies for the preservation
and maintenance of spermatozoa, and it is broadly applied
in malignancies or other therapies which could damage to
the functionality of testicles (
Many efforts have been made to minimize the rate of cryodamage in frozen spermatozoa during
the cryopreservation process. For instance, the addition of antioxidant agents or
cryoprotectants to the extender is one of the promising tools for the increase of sperm
quality during the freeze-thaw process (
Hence, regarding the above statements, it would be plausible that LIPUS can enhance the
penetration of herb extraction into the sperm via an increase in motility of spermatozoa.
Therefore, the primary goal of the present study was to determine the impact of
In this experimental study, we evaluated the effects of
μg/ml (CGM group), frozen-thawed spermatozoa exposed to the US radiation (LIPUS-exposed group) at a duty cycle of 40% (pulsed mode, at a frequency of 1 MHz, at the incident power density of 200 mW/cm2 for 200 seconds), and frozen-thawed spermatozoa treated with the combination of Calligonum extract and the US radiation with continues mode (CGM+LIPUS group). The present research was approved by Ethical Committee of Tarbiat Modares University (No. 52/6757 dated 30.11.92).
The plant (
Two kilograms of the fresh aerial parts of the plant (equal to 50% of the weight of a wildly-growing plant) were air-dried in the shade at room temperature, grounded, and exhaustively extracted by cold maceration with aqueous methanol (70%). The extract was evaporated under reduced pressure at 40°C to yield 80 g residue. The residue was suspended in distilled water and successfully fractionated with n-hexane, CH2Cl2, EtOAc, and n-BuOH (Thermo Fisher, USA) saturated with H2O. Each extract was evaporated under reduced pressure to yield 3, 7, 12 and 22 g residues, respectively.
The activity of free radical-scavenging of the methanolic extract of C. comosum was
determined concerning the potential to neutralize the free radical-producing
[2,2-diphenyl- 1-picrylhydrazyl (DPPH)] according to a method, as described previously
In this experimental study, semen samples were obtained from twenty-five fertile men with
the average age of 34 (range,
A fraction of motile spermatozoa was selected to be analyzed by the swim-up method. For
this purpose, 1 ml of each semen sample (kept at 37°C and 5% CO2) was added to
3 ml of human tubal fluid (HTF, Genocell Co., Iran) supplemented with 10% human albumin
serum (HAS, Biotest, Germany) and centrifuged at 2000 ×g for 3 minutes. Afterward, 0.5 ml
of HTF supplemented with 10% HAS was gently added on the resultant pellets. The samples
were then incubated at 37°C, 5% CO2, and inclined at a 45° angle to incubator
for 45 minutes. Consequently, 0.5 ml of the uppermost medium was recovered, and the
swim-up method was performed (
Each processed semen sample was cryopreserved according to the standard protocol for
sperm freezing. According to Li et al. (
At the thawing stage, the cryo-straws were removed from liquid nitrogen and immediately
immersed in a water bath at 37°C for at least 1-2 minutes. The thawed straws containing
semen samples were flicked and inverted to mix the contents before sampling thoroughly and
then washed with a culture medium HTF supplemented with 10% HAS (The samples were then
The US device (Physiomed, Germany) was set up at the frequency of 1 MHz, incident power
density of 200 mW/cm2, 200-second time period, and a 45-minute period as the
duration of the experiment. These fine-tuned parameters were chosen based on our previous
study published in this regard (
Sperms maintained in the HTF medium supplemented with 10% HAS (Gibco, Germany). The samples were exposed to low-intensity pulsed ultrasound (LIPUS) (1 MHz, 200 mW/cm2 and 40% DC) alone or in combination with C. comosum coined as experimental groups. The control group was also cultured in the HTF medium supplemented with 10% HAS. After the US stimulation, spermatozoa were incubated for 45 minutes in 5% CO2 at 32˚C ,similar to other experimental groups. To investigate the sperm parameters, the mean number of whole cells per volume, viability, morphology, and motility were examined after the incubation process.
The sperm count, motility, morphology, viability, and
DNA fragmentation were evaluated according to the
guidelines introduced by the WHO (
The Halotech DNA G2 kit (Spain) was used to study the DNA fragmentation in frozen-thawed spermatozoa. Based on the sperm chromatin dispersion test (SCD), the intact frozen-thawed spermatozoa were diluted in a culture medium HTF supplemented with 10% HAS to achieve sperm concentration of 15-20 million per milliliter. In this method, 50 μl of the semen sample was added to 100μl of dissolved agarose (0.7%) (Sigma, US). Afterward, 25 μl of the cell suspension was transferred to slides, pre-coated with 0.65% agarose and covered with a coverslip without any air bubbles, and then incubated at 4°C for 5 minutes. After the removal of coverslips, the slides were horizontally immersed in a tray with freshly prepared acid denaturation solution (0.08 N HCl) for 7 minutes at 22°C in a dark place to create restricted single- stranded DNA from DNA breaks. The slides were then immersed in lysis Solution I [0.4 M Tris (Sigma, USA), 0.4 M 2-Mercaptophenol (Sigma, USA), 1% sodium dodecyl sulfate (SDS, Sigma, USA), and 50 mM Ethylenediaminetetraacetic acid (EDTA, Sigma, USA), pH=7.5 for 20 minutes and lysis Solution II (0.4 M Tris, 2 M NaCl, and 1% SDS, pH=7.5) for 5 minutes to remove nuclear proteins. Slides were then rinsed with distilled water for 5 minutes, followed by dehydration through an ascending gradient of ethanol (70, 90, and 100%) for 2 minutes. The slides were then placed at room temperature to be air-dried.
For Diff-Quik staining, the slides were incubated in eosin solution for 6 min; then, transferred into Azur B solution, for another 6 minutes. The nucleoid of spermatozoa with fragmented DNA did not develop a dispersion halo, or the halo was minimal. From each slide, a minimum of 500 spermatozoa was scored under an oil-immersion objective (×100 magnification) by light microscopy (Labomed, USA). The sperm cells showing no halo, small halo, medium halo, large halo, or fragmentation were separately scored. Spermatozoa indicating no halo/fragmentation were considered to have damaged chromatin, and the results were expressed as a percentage of sperm cells with DNA damage.
Spermatozoa were rinsed with phosphate-buffered saline
(PBS, Atocel, Austria) and incubated with 20 μM 2,
7- dichlorofluorescein diacetate (DCFH-DA, Life Technologies,
USA), diluted in serum-free medium at 37°C for
45 minutes. The intracellular ROS level was immediately
analyzed with flow cytometry at an excitation wavelength
of 488 nm and an emission wavelength of 530 nm (
The analysis of the values obtained in this study was performed by the SPSS version 19 (SPSS Inc., IBM company, USA), while the level of significance was set at P<0.001. The difference between the values of each group was analyzed by one-way analysis of variance (one-way ANOVA), followed by Tukey’s test. The data were expressed as the means ± standard deviation (means ± SD).
The demographic characteristics of patients are demonstrated in Table 1.
The demography of semen samples
|Total motility (%)||80.7795||1.69|
|Grade A (%)||33.1496||1.74|
|Grade B (%)||30.7443||1.56|
|Grade C (%)||16.8856||1.39|
|Grade D (%)||19.4599||1.58|
|Grade A+B (%)||64.0651||2.08|
|Normal morphology (%)||28.6397||4.26|
Grade A; Linear progressive motility, Grade B; Progressive motility, Grade C; Non-progressive motility, Grade D; Immotile, SDF; Sperm DNA fragmentation, ROS; Reactive oxygen species, and SD; Standard Diviation.
All of the sperm parameters, including viability, motility, and morphology in all
experimental groups, are shown in Table 2. Accordingly, the percentage of total motility
of spermatozoa in the fresh group was 89.37 ± 3.74, while this value was 81.30 ± 5.72 in
the frozen-thawed group. After the addition of 10 μg/ml of Calligonum extract in the
freezing medium increased the total motility; yet, such an increase was not statistically
significant when compared with the control group (P≥0.05). LIPUS (in pulsed and continues
mode waves) alone decreased the motility of spermatozoa, compared with the control group
(P≤0.038), but the combination of
As shown in Table 2, the viability of spermatozoa in all groups undergone the freeze-thawing process was significantly reduced as compared with the fresh group (P≤0.026). Such a reduction was more pronounced in the LIPUS-exposed and CGM-treated groups, compared with other groups. There was no significant difference between the CGM+LIPUS and control groups (frozen-thawed spermatozoa) (frozen-thawed spermatozoa, P≥0.05).
Comparison of sperm parameters (± SD) between the experimental groups after frozen-thawed and treatment with 10 μg/ml Calligonum (CGM) extract and LIPUS (pulsed mode and continues wave)
|GroupsSperm parameters||Control||Frozen -thawed||Frozenthawed+CGMextract||Frozenthawed+continueswave of ultrasound||Frozenthawed+continueswave oftrasound+CGMextract||Frozenthawed+pulsedmode ofultrasound||Frozenthawed+pulsedmode of ultrasound +CGMextract|
|Viability (%)||94.17c ± 5.97||87.52 ± 4.75||90.11 ± 6.12||82.76b ± 5.68||86.70 ± 7.12||81.82b ± 2.87||87.94 ± 4.25|
|Total motility(%)||89.37 ± 3.74||81.30 ± 3.74||85.42 ± 4.28||76.76ab ± 5.29||81.65 ± 5.18||75.86ab ± 3.47||81.90 ± 3.93|
|Progressivemotility (%)||38.80 ± 2.57||34.81a ± 3.28||37.81 ± 4.13||33.04ab ± 2.98||37.24 ± 5.11||32.60ab ± 3.58||36.18a ± 4.78|
|Normalmorphology (%)||30.11 ± 3.16||26.58 ± 3.54||28.41 ± 4.27||27.58 ± 3.95||30.82 ± 2.75||28.35 ± 4.82||30.05 ± 3.66|
a; Significant difference with control group in the same row (P≤0.05), b; Significant difference with freeze and thawed group + CGM extract, in the same row P≤0.026, and c; Significant difference with other groups in the same row (P≤0.047).
Assessment of fragmented DNA and free radicals percentage in all experimental groups
|Groups||Control||Frozen- thawed||Frozen- thawed +CGM extract||Frozen- thawed +continues wave ofultrasound||Frozen- thawed +continues wave ofultrasound+CGMextract||Frozen- thawed +pulsed mode ofultrasound||Frozen- thawed +pulsed mode ofultrasound+CGMextract|
|Fragmented DNA(%) ± SD||24.5 ± 7.23||46.5a ± 9.26||37 ± 4.29||38 ± 5.37||34 ± 5.17||51a ± 11.85||42.5a ± 7.39|
|Freeradicals(ROS)(%) ± SD||-||12.46 ± 7.06||9.18 ± 2.57||12.52 ± 8.14||7.77 ± 2.06||14.75 ± 4.67||10.05 ± 1.68|
CGM; Calligonum extract, ROS; Reactive oxygen species, and a; significant difference with control group in the same row (P≤0.042).
According to Table 2, the normal morphology score was 30.11 ± 3.16 in the fresh group, while the normal morphology score was 26.58 ± 3.54 i the control group. The statistical analysis revealed that there was no significant difference in the score of normal morphology among all treated groups, namely, the CGM-treated, LIPUS-exposed, and CGM+LIPUS groups.
Table 3 shows the rate of DNA fragmentation in all experimental groups. The percentage of spermatozoa undergone DNA fragmentation was considerably higher in all groups than the fresh group. The rate of DNA fragmentation was significantly (P≤0.042) elevated in the frozenthawed (46.5 ± 9.26) and LIPUS-exposed groups (51 ± 11.85) in comparison with the control group.
According to Table 3, the level of ROS in the control group receiving no treatment was 12.46 ± 7.06, while the level of ROS was decreased to 9.18 ± 2.57 in the CGM group. The statistical analysis demonstrated that the difference in the amount of ROS was not significant in all experimental groups as compared with the control group (P≥0.05).
Cryopreservation of human semen provides a valuable
therapeutic opportunity for the management of patients
who are at risk of infertility (
It has been implicated that antioxidant therapy can increase the quality of cryopreserved
spermatozoa when employed both
Our data revealed
On the other hand, LIPUS at the frequency of 1 MHZ and incident power density of 200
mW/cm2, in both pulsed and continuous mode waves, had adverse impacts on the
sperm parameters. In the LIPUS-exposed group, the viability, as well as total and
progressive motility was decreased while the number of none-motile spermatozoa was increased
as compared with the fresh group. The US radiation alone increased intracellular ROS level
and disrupted the balance of pro-oxidant and antioxidant contents in human spermatozoa, led
to the elevated rate of DNA fragmentation. Also, our data demonstrated that LIPUS did not
alter the morphology of frozen spermatozoa; however, the combinatory treatment of
Previous studies have highlighted that LIPUS, as mechanical
energy, could have therapeutic effects on bone
and wound healing (
In the present study, the adverse effects of LIPUS on the
sperm parameters may be due to the changes in the frequency
of waves as a result of the sperm motility, leading
to the reduction in the effectiveness of LIPUS penetration.
Since the distribution of the US field is not homogenous homogenous,
and it is susceptible to the reflection and attenuation
once the field passes through the boundary separating
two different media (
Our results showed that