The Effects of Calligonum Extract and Low-Intensity Ultrasound on Motility, Viability, and DNA Fragmentation of Human Frozen-Thawed Semen Samples

In this experimental study, we evaluated the effects of Calligonum extract and LIPUS at a frequency of 1 MHZ (in pulsed and continues wave modes) on the cryopreservation cryopreservation of human spermatozoa. After the preparation of semen samples, each sample was liquated into 5 parts included; washed spermatozoa (control group), frozen-thawed spermatozoa, frozen-thawed spermatozoa treated with Calligonum extract at a concentration of 10

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 (Calligonum comosum L.) was collected by Dr. Hosein Batooli from the desert located at the proximity of Kashan, Iran, (33.9850°N, 51.4100°E). The taxonomic identity of the collected plant was confirmed by Dr. Abdoalrasool Haghir Ebrahim Abadi, Faculty of Science, Kashan University, Iran. Freshly collected plant materials were air-dried in the shade at room temperature. The aerial parts (stem, flowers, and leaves) of this herb were used for further investigations.

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 (19, 20). Concisely, the level of DPPH was calculated by the measurement of the absorbance at the wavelength of 517 nm prior to and after the addition of a specific amount of the herb extract. Afterward, the inhibitory percentage of radical formation was estimated using the following equation: % inhibition= (A _control – A _sample)/A _control ×100, where A _control is considered the absorbance of DPPH alone and A _sample is regarded as the absorbance of DPPH in combination with the C. comosum extract.

In this experimental study, semen samples were obtained from twenty-five fertile men with the average age of 34 (range, 25-50 years) who referred to the in vitro fertilization (IVF) center of Gandhi Hospital according to the World Health Organization (WHO) criteria. The process of semen collection was carried out by masturbation into a sterile, wide-mouthed, calibrated glass container after five days of sexual abstinence. The collected specimens were allowed to liquefy at 37°C for 30 minutes prior to the semen analysis. The entire samples were collected from individuals who referred for medical procedures during assisted reproductive technology (ART). We used the remaining samples obtained from patients, with their permission, for research purposes.

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 (21).

Each processed semen sample was cryopreserved according to the standard protocol for sperm freezing. According to Li et al. (7) and Ibrahim et al. (22), semen samples were gently mixed with an equal volume of modified cryoprotective medium (Global Media, USA) supplemented with 10 μg/ml of Calligonum extract. The samples were kept at 4°C for 30 minutes and then frozen by placing the straws horizontally at 10 cm above the surface of liquid nitrogen (nitrogen vapor) at -80°C for 20 minutes. Finally, all frozen straws were stored in liquid nitrogen until use.

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 treated with Calligonum extract (10 μg/ml)). At the end step, semen samples were centrifuged at 2000 ×g for 3 minutes to remove any trace of cryoprotectant in the freezing medium. The samples were analyzed for motility, viability, morphology, and DNA fragmentation (9, 23).

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 (24). The US stimulation was carried ou in which the transducer was put at the focal distance (3.5 cm) of a cell culture plate incubated in 5% CO₂ at 32˚C. It was transmitted through the bottom of the well via coupling gel between the transducer and the tube. The device was transmitted through the bottom of the well via coupling the gel between the transducer and cell culture plate. Notably, no temperature change more than 1ºC was recorded during the US stimulation.

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 (25). The sperm motility analysis was conducted using light microscopy (at ×400 magnification), combined with a computer-assisted semen analysis system (CASA, Video Test Sperm 1.2, Russia). A Makler chamber was used for the scoring of the sperm motility at room temperature. A minimum of 100 spermatozoa from at least five different fields was analyzed from each aliquot. Sperm morphology was assessed by the Diff-Quik staining method according to WHO guidelines by light microscopy (Labomed, USA). The sperm viability was determined by Eosin B (5% in saline) (Merck, Germany) staining technique at onestep. In this procedure, viable or dead spermatozoa are recognized by white (unstained) and pink (or red) coloration in the head region of the sperm cells, respectively.

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 (26).

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 C. comosum extract showed free radical-scavenging activity in a dose-dependent manner when examined by the DPPH assay. The inhibitory concentration (IC₅₀) value (the concentration of a given substance causing 50% inhibition of the DPPH) of the herb extract was 29.2 μg/ml when compared with ascorbic acid (positive control).

The demographic characteristics of patients are demonstrated in Table 1.

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 Calligonum extract and LIPUS increased the motility to (81.90 ± 3.93) and (81.65 ± 5.18) respectively when used in pulsed mode and continuous modes waves. Also, there was an improvement in progressive motility of spermatozoa treated with the combination of Calligonum extract and LIPUS, compared with the frozen- thawed group; however, the difference between two groups was not statistically significant (P≥0.05).

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).

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).