The effect of low-power laser on the promotion of spermatogenesis in a mouse model of azoospermia (in-vivo)
habib tajalli
1
(Biophotonics Research center)
Masoud Maleki
2
(Department of Basic Science, Biotechnology Research Center, Tabriz Branch, Azad Islamic University, Tabriz, Iran)
Esmail Safavi
3
(Department of Basic Sciences, Faculty of Veterinary Medicine, Tabriz Branch, Azad Islamic University, Tabriz, Iran)
Reza Shahi
4
(Department of Mathematics and Statistics, Tabriz branch, Islamic Azad University, Tabriz, Iran)
Fatemeh Firoozi
5
(Department of Basic Science, Biotechnology Research Center, Tabriz Branch, Azad Islamic University, Tabriz, Iran)
Zahra Akbarpour
6
(Department of Basic Science, Biotechnology Research Center, Tabriz Branch, Azad Islamic University, Tabriz, Iran)
Ali reza Sotoudeh Khyaban
7
(Research Center of Biophotonics, Islamic Azad University, Tabriz Branch, Tabriz, Iran)
الکلمات المفتاحية: Infertility, Spermatogenesis, azoospermia, Laser therapy, Busulfan, low power laser,
ملخص المقالة :
This research aimed to investigate the effect of low power laser on spermatogenesis in testicular tissue of azoospermia mouse model in-vivo. In this experimental work, 112 adult male Syrian mice were randomly divided into three groups (harboring 8 mice each): negative control, positive (Azoospermia control) group, and experimental group. To create azoospermia in positive animals, Busulfan was used at a dose of 30mg/kg, for 21 days by intraperitoneal injection. In the experimental group after Busulfan treatment, they were affected by low power diode laser (wavelength of 808nm) at three different energy densities of 2, 4, and 8 J/cm2. The employment of a laser with an energy density of 8 J/cm2 was shown to be beneficial in boosting germ cell and sperm production.
The effect of low-power laser on the promotion of spermatogenesis in a mouse model of azoospermia (in-vivo)
Habib Tajalli1, 2*, Masoud Maleki3, 4, Esmail safavi4, 5, Reza Shahi6 , Fatemeh Firouzi4
Zahra Akbarpour4, Alireza Sotoudeh1, 2
1-Biophotonics Research Center, Tabriz Branch, Azad Islamic University, Tabriz, Iran
2-Department of Physics, Tabriz Branch, Azad Islamic University, Tabriz, Iran
3-Department of Biology, Tabriz Branch, Azad Islamic University, Tabriz, Iran
4-Department of Basic Science, Biotechnology Research Center, Tabriz Branch, Azad Islamic University, Tabriz, Iran
5-Department of Basic Sciences, Faculty of Veterinary Medicine, Tabriz Branch, Azad Islamic University, Tabriz, Iran
6-Departement of Mathematics, Tabriz Branch, Azad Islamic University, Tabriz, Iran
Abstract
This research aimed to investigate the effect of low power laser on spermatogenesis in testicular tissue of azoospermia mouse model in-vivo. In this experimental work, 112 adult male Syrian mice were randomly divided into three main groups (harboring 8 mice each): negative control, positive (Azoospermia control) group, and experimental group, but to determine the best dose of laser radiation several experimental groups were tested. To create azoospermia in positive animals, Busulfan was used at a dose of 30mg/kg, for 21 days by intraperitoneal injection. In the experimental group after Busulfan treatment, they were affected by low power diode laser (wavelength of 808nm) at three different energy densities of 2, 4, and 8 J/cm2. The employment of a laser with an energy density of 8 J/cm2 was shown to be beneficial in boosting germ cell and sperm production.
Keywords: spermatogenesis, azoospermia, laser therapy, infertility
Introduction
Infertility is described as a man or woman's failure to conceive following 12 months of unprotected sexual activity. This condition affects up to 15% of the world's population, with a male factor accounting for 50% of cases (1-3).
Male infertility is a multifactorial disorder that encompasses a wide variety of diseases and is a sign of a wide range of pathological conditions affecting both the sexual and other physiological systems, including the endocrine, neurological, blood and immunological systems (4-6).
According to the recommendations of the World Health Organization (WHO) (2000), 16 main nosologies are distinguished, each of which, in turn, includes upwards of several dozen specific pathogenetic factors, 4 of 16 diagnoses are descriptive, without indicating the true cause: idiopathic oligo-, astheno-, terato- and azoospermia (7, 8).
Diseases such as cancers of the reproductive system in young men, infections with bacterial infections, some genetic syndromes such as praderovirus (PWS), and mutations in fertility genes can cause azoospermia in men (9). Azoospermia means the lack of sperm in the semen. One percent of all men and ten percent of infertile men have azoospermia. In many of these men, there are no recognizable signs of semen appearance. The volume and shape of semen are normal and the majority of these men have normal sexual desire and sexual function and only have fertility problems (10). There are several factors involved in the development of azoospermia. Non-obstructive genetic azoospermia is caused by hormonal and genetic disorders, trauma to the testicles and blood vessels, varicocele, and so on. Factors that may cause obstructive azoospermia to include vasectomy, congenital absence of the seminal vesicles, obstruction of the epididymis for unknown reasons, or acquired epididymal obstruction, for example, due to infections, especially sexually transmitted infections, and some inherited diseases such as Cystic fibrosis (CF) noted (11).
Today, various methods are used to treat infertility, including IUI1, IVF2, ICSI3, and laser therapy. However, each of these methods is used for a specific range of infertile people (12).
Laser therapy, which is a modern method of physiotherapy, is an example of interdisciplinary medicine based on research in the fields of physiology, biophysics, and biochemistry, and effectively offers treatment techniques according to the patient's personal and individual conditions and characteristics (13). Common methods used to improve and increase fertility in animals and humans are based on the use of drugs or direct injection of sperm into the egg, which is not always effective (14). Laser light irradiation is an effective alternative to conventional syringe injection because, in addition to being safe and non-invasive, it is also a suitable and practical method for patients who are afraid of syringes (15).
Laser therapy or low-level laser therapy (LLLT) is widely used in modern medicine due to its high efficiency, ease of use and lack of side effects is a natural and biological tissue effect with the help of energy and light power (13, 16).
It is consisting of the application of light in continuous or pulsed wave modes within the near-infrared range (600 to 1100 nm) in an attempt to promote tissue repair and analgesia and reduce inflammation (17). It is a non-invasive remedy that employs energy densities and wavelengths that may effortlessly penetrate tissues, ensuing in photobiomodulation results on cells and tissues of numerous layers(18). Indwelling tissue, the laser develops new systems and responses (16).
The use of different mechanisms of laser in human and animal cell culture and at the invivo condition that elicits responses at the molecular, cellular, and tissue level and results in cell proliferation and tissue repair have been reported in the literature, including an increase in metabolism, mitochondrial activity, proliferation, migration, adhesion, differentiation, extracellular matrix secretion, and mineralization, as well as the inhibition of apoptosis (19-22). Its primary mechanism of action is the stimulation of the mitochondrial respiratory chain and the beginning of cellular signaling, which is a wavelength, irradiance, and dose-dependent (23).
The specific performance of laser irradiation on sperm has been investigated in animal models. In these studies, which used lasers with different powers, different radiation energies, and different radiation durations, the results showed that the use of lasers with low power and low energy density has the most appropriate effect on the quantitative and qualitative parameters of sperm. For example, a study by N. Laffaldano et al. the quality of turkey sperm cells showed that exposure to radiation at 3 J/cm2 centimeters increased the survival rate of turkey sperm compared to controls (24). In another study by Corral-Baqués MI et al., dog sperm cells were subjected to laser irradiation at some energy density and results showed that Irradiating dog sperm with a 655-nm continuous-wave diode laser at 4, 6 and 10 J/cm2 energy density doses improves its motility features and seem to maintain its functional characteristics (25).
The effects of Low-Level Light Therapy (LLLT) on cellular function arise predominantly from stimulation of ATP production and reduction of oxidative stress. These effects are dose-dependent and a function of beam irradiance and irradiation time. Human sperm motility has been shown to increase with LLLT irradiation (26). In 2018, C. Philip Gabel et al. Examined the effect of low-level laser therapy on the function and motility of human sperm and the DNA integrity of these cells. Because the effects of lasers depended on the amount of radiation and irradiation time, the results showed a four-fold increase in ATP production in spermatozoa and their movement compared to the control group (26).
These findings also provide insights into the effect of laser light on increased fertility in humans. A 2012 study by Ross S. Firestone examined the effects of low-level laser light on sperm motility and DNA damage. Collected human specimens were classified as normozoospermic, oligospermia, or asthenospermic. The samples were treated with a 30-second pulse of a 905 nm and 30 MW infrared laser, which appears to increase the optical sensitivity of cytochrome c oxidase in the electron transport chain in the mitochondria. A significant increase in motility was most characteristic in oligospermia and asthenospermic specimens observed after treatment but no increase in DNA damage was observed compared to control specimens (27).
Although laser therapy is beneficial in the treatment of many tissue damages, little is known regarding its effect on the testis and spermatogenesis. The proper development of germ cells depends on the proper functioning of all cells in the testicular tissue. Seminiferous tubules, Sertoli, and Leydig cells make up the testis, which produces sperm and testosterone. Sperm cells are generated during a process that calls spermatogenesis. Stem cell spermatogonia, which produces spermatozoa through a series of cell division and differentiation processes known as spermatogenesis, is very sensitive to radiation. When spermatogonia are exposed to radiation, DNA damage occurs, resulting in cell cycle arrest. If the level of DNA damage is severe enough, the entire population of spermatogonia may perish, resulting in infertility (28). Although some studies showed that several types of spermatogonia are resistant to radiation (29-31). On the other hand, biochemical and topological analyzes show that spermatozoa exposed to laser have high fertility (32). Therefore, the production of sperm may vary depending on the radiation dose and dose rate.
Due to the importance of fertility in men and the problems caused by azoospermia that cause infertility, this study was performed to determine the effect of low energy laser beam density on seminiferous tubules and spermatogenesis in testicular tissue of in-vivo azoospermic mice.
Materials and Methods
In this experiment, adult male mice weighing approximately 35+/-5 g were kept in suitable temperature conditions (22+/-2 ° C) and 12 hours of light, and 12 hours of darkness with the same water and food conditions and without restrictions, were used.
At this stage of the study, 112 mice were randomly divided into 3 groups: negative control, positive control (azoospermia control), and experimental (in 3 groups) (n = 8). Negative control animals were not treated under any conditions. To create azoospermia in positive control animals, Busulfan was used at a dose of 30mg/kg (33, 34), 21 days by intraperitoneal injection. In the experimental groups after Busulfan treatment, they were affected by low-power diode lasers at three different energy densities.
The experimental groups were exposed to a diode laser (wavelengths 808nm) with different energy densities (2, 4, 8 J/cm2). The area in the left testes of mice to be irradiated was shaved and cleaned with alcohol before each treatment. The light beam was applied once every two days for 21 days.
In the next step, the animals of the negative control, positive control (azoospermic control), and experimental groups were killed by ether anesthesia after 40 days, and sampling was performed on their testes using histomorphometric measurement. The testes were prepared in tissue sections.
Finally, the prepared sections were stained, and microscopic studies were performed on negative control, positive control, and experimental samples by morphological criteria. Microscopic images were evaluated using software Cellness.
In each group, the diameter of the seminiferous tube, the diameter of the epithelium, the area of the epithelial cell in the seminiferous tubule, and the diameter of the seminiferous duct were measured and statistically compared with magnifications 40x, 100x and 200x. For all groups, mean and standard deviation were calculated. Data were analyzed by one-way ANOVA tests with software SPSS. The p-value was considered less than 0.05.
[1] Intrauterine insemination
[2] In vitro fertilisation
[3] Intracytoplasmic sperm injection
Results
Morphological findings of testicular tissue in the studied groups
Seminal vesicle degradation and a significant reduction in the number of germinal epithelial cells are seen in the testicular histology of the azoospermic group. A relatively limited number of spermatogenesis cells are observed, which are mainly spermatogonial cells. Interstitial tissue has developed and the diameter of the seminiferous tubules has decreased. The wall of most seminiferous tubules is empty of spermatogenesis cells (Figure1). An increase in the thickness of the epithelial tissue of the tubes was detected in the groups that are exposed to the laser at 2 J/cm2 and 4 J/cm2, and repair in these tubes was seen initially (Figure 2,3). A small number of spermatogenesis cells are found in the walls of the seminiferous tubules. In the group that is exposed to the laser at 8 J/cm2, the process of testicular tissue repair was faster, and the diameter of the tubes and the thickness of the tubular epithelium increased, which indicates the improvement of pathological testicular lesions and also the number of germ cells in its wall increased (Figure 4).
Histomorphometric findings of testicular tissue:
The diameter of the seminiferous tubules and the thickness of the epithelium of the tubes in the groups that received the laser at 2 J/cm2 and 4 J/cm2 showed a significant increase compared to the azoospermia group (P <0.05). However, there was no significant difference between the 2 J/cm2 and 4 J/cm2 groups (Figure 5). In the group receiving the laser at 8 J/cm2, the diameter of the seminiferous tubules and the thickness of the epithelium of the tubes showed a significant increase compared to the 2 J/cm2 and 4 J/cm2groups (P <0.05) (Figure 6).
Examination of interstitial tissue thickness in different groups showed that the groups received irradiation had a significant decrease compared to the azoospermic group, but no significant difference was observed between the groups with different energy densities (Figure 7).
Testicular spermatogenesis findings:
Evaluation of T.D.I coefficient in different groups showed that the regeneration coefficient in the groups that received the laser at 2 J/cm2 and 4 J/cm2compared to the azoospermia group increased significantly (P <0.05). But there is no significant difference between the 2 J/cm2 and 4 J/cm2groups. The group receiving laser at 8 J/cm2, showed a significant increase compared to the groups of 2 J/cm2 and 4 J/cm2 (P <0.05) (Figure 8).
Evaluation of the S.I. coefficient in different groups did not show a significant difference between the groups (Figure 9).
On the other hand, the evaluation of R.I in different groups showed that the group received the laser irradiation at 8 J/cm2 there was a significant increase compared to the other groups (Figure 10).
A comparison of the mean of evaluated parameters in the studied groups is shown in Table 1
Figure 1. Microscopic view of testicular tissue in azoospermia group: reduction of seminiferous tubules diameter and severe reduction of tubular epithelial thickness is observed. Spermatogenesis cells are found in very small numbers in the walls of the tubes. (40x,100x and 200x magnification, hematoxylin-eosin staining)
Figure 2. Microscopic view of testicular tissue in azoospermia group with 2J/cm2 energy density irradiation: Testicular tissue repair is initially evident, increase in seminiferous tubule diameter and tubular epithelium repair are seen partly. The number of spermatogenesis cells has increased slightly. (40x,100x and 200x magnification, hematoxylin-eosin staining)
Figure 3. Microscopic view of testicular tissue in azoospermia group with 4J/cm2 energy density irradiation Testicular tissue repair is initially evident, increase in seminiferous tubule diameter and tubular epithelium repair are seen partly. The number of spermatogenesis cells has increased. (40x,100x and 200x magnification, hematoxylin-eosin staining)
Figure 4. Microscopic view of testicular tissue in azoospermia group with 8J/cm2 energy density irradiation: Testicular tissue repair is evident, increase in seminiferous tubule diameter and tubular epithelium repair are seen. The number of spermatogenesis cells has increased sharply. (40x,100x and 200x magnification, hematoxylin-eosin staining)
Thickness (µm) |
Dose of energy of laser irradiation |
Figure 5. Comparison of mean ± standard deviation of seminiferous tubular epithelial thickness in different groups
*#@: Different signs indicate significant differences between groups.
Diameter (µm) |
Dose of energy of laser irradiation |
Figure 6. Comparison of mean ± standard deviation of seminiferous tube diameters in different groups
*#@: Different signs indicate significant differences between groups.
Thickness (µm) |
Dose of energy of laser irradiation |
Figure 7. Comparison of mean ± standard deviation of interstitial tissue thickness in different groups
*#: Different symbols indicate significant differences between groups.
Tubular differentiation index (%) |
Dose of energy of laser irradiation |
Figure 8. Comparison of mean ± standard deviation of T.D.I coefficient in different groups
*#@: Different signs indicate significant differences between groups
Sperm production index (%) |
Dose of energy of laser irradiation |
Figure 9. Comparison of mean ± standard deviation of S.I. coefficient in different groups
*#@: Different signs indicate significant differences between groups.
Reproductive index (%) |
Dose of energy of laser irradiation |
Figure 10. Comparison of mean ± standard deviation of R.I coefficient in different groups
*#: Different symbols indicate significant differences between groups.
Table 1. Comparison of the mean of the evaluated parameters in the testicular tissue of the studied groups
Azoospermic group+4J/cm2 irradiation | Azoospermic group+ 2J/cm2 irradiation | Azoospermic group | parameter | |
40.54±6.14c | 21.60±2.89b | 20.67±3.43b | 2.85±2.30a | Epithelial thickness (micrometers) |
163.27±6.16c | 149.99±10.51b | 145.83±9.14b | 119.09±12.43a | Diameter of seminiferous tubules (micrometers) |
10.48±2.75b | 13.84±3/13b | 14.47±3.02b | 18.71±5.78a | Interstitial tissue thickness (micrometers) |
26.30±3.89c | 12.60±2.32b | 12.20±2.30b | 1.20±0.92a | T.D.I.1 |
1.10±088a | 1.00±0.67a | 1.00±0.67a | 0.40±0.70a | S.I.2 |
24.70±7.86b | 7.00±2.06a | 5.40±2.12a | 2.40±1.18a | R.I.3 |
Different abc letters in each row show a significant difference (p <0.05)
[1] Tubular differentiation coefficient
[2] Spermiogenesis coefficient
[3] Reconstruction coefficient
Discussion
This study, which aimed to evaluate the effectiveness of laser light on male infertility, showed that using the right dose of laser can be a good treatment for Busulfan-induced azoospermia. According to other reports (33, 34), our findings confirm that Busulfan at an effective dose of 30mg/kg reduces the number of sperm cells to create an azoospermic model. However, in this study we showed three experimental groups exposed to laser radiation with a density of 2,4,8 J/cm2, all three groups presented satisfactory results of testicular repair compared to the group treated with Busulfan.
Busulfan is a chemotherapy drug used to treat some diseases such as leukemia, although it has been associated with male infertility. It is a powerful chemical agent that preferentially destroys spermatogonia stem cells and disturbs the spermatogenesis process by affecting germ cells and sertoli cells (35). The morphological and histomorphological findings of the azoospermic control group confirmed previous researches (36, 37), showing substantial atrophy of the seminal vesicle, a severe decrease of reproductive epithelial cells, and the presence of a very small number of spermatogonia cells. The testes in this azoospermia model were dwindled by Busulfan.
Many investigations have been conducted so far to modify and abolish the infertile effect of Busulfan. In a study, the therapeutic effect of bone marrow-derived mesenchymal stem cells on Busulfan-induced azoospermia demonstrated that the seminiferous tubules' epithelial tissue was normal and that spermatogenesis was detectable in the majority of these tubes. These stem cells, which have differentiation potential, are a good choice for therapeutic applications due to the secretion of anti-inflammatory cytokines and growth factors (38) . In another study, a condition medium from these stem cells was used to treat Busulfan-induced azoospermia. Histomorphological findings showed an increased thickness of the azoospermic seminiferous tubules compared to the control group (37). Overall, these studies show that the anti-infertility effect of Busulfan is treatable and provides a way to use easier and less risky treatments such as photobiomodulation.
Today, photobiomodulation is widely used in the treatment of infertility disorders. According to other reports that have been presented so far about the use of laser therapy in the treatment of infertility (8, 26, 32), our results also confirmed the restorative effect of low-power laser on testicular tissue. In this study, which used a diode laser with a wavelength of 808nm and continuous radiation, three experimental groups were affected by three different doses of energy, namely 2, 4, and 8 J/cm2. Histological criteria and testicular spermatogenesis showed a significant change in experimental groups compared to the Busulfan control group and spermatozoa cells were well detectable after laser efficacy. According to previous studies, light modulation can increase cellular metabolism after receiving energy and modulate cellular processes such as proliferation, differentiation, and tissue repair (8). Therefore, energy and optical power, if given in the right dose, will have a natural and biological effect on the tissue and lead to the initiation of some reactions in the living cell (39). In our study, comparing the effectiveness of three doses of energy, the energy dose of 8 J/cm2 was more efficient compared to the doses of 2 and 4 J/cm2, which means that under the influence of a higher energy dose, testicular volume was increased and the presence of germ cells in the seminiferous tubules were observed after extensive degradation due to the effect of Busulfan, which showed stimulation of spermatogenesis and balance between proliferation and cell differentiation.
There are still unanswered questions about what range of wavelength lasers and in what range of power and radiation energy can have the most effect. In general, studies have shown that light modulation using laser can increase sperm motility (24-27). These studies showed that laser light stimulates the production of ATP and reduce oxidative stress, leading to increased sperm motility. Laser light affects the bipolar lipid layers of the membrane and thus the ion channels for the transport of calcium and other ions, altering the permeability of the mitochondrial membrane and activating the electron transfer chain, which ultimately leads to an increase in ATP and Produces the energy needed for sperm motility (40). For example, Daryl Precee and colleagues 2017 (14) measured the motility and chromatin structure of sperm using a 633nm red-wave He-Ne laser with a radiation power of 31mW/cm2. They found that laser radiation increases sperm motility. However, this amount of radiation power causes damage, albeit minor, to the DNA structure. Therefore, the primary importance in the therapeutic application of laser is to investigate the lack of damage to the DNA structure due to laser light irradiation (14). In a recent study, the results show that the Ga.Al.As infrared laser with a wavelength of 808nm and an energy density of 28 J/cm2 not only did not affect increasing testosterone concentration, sperm quality, and motility but also caused the appearance of lesions in the sperm (41). The reason for this result is that increasing the radiation dose from an acceptable level, by over-stimulating aerobic phosphorylation leads to an unregulated increase in ROS production and oxidative DNA damage resulting in cell death due to oxidative stress (42, 43). According to the Moskvin paper, the kinetics of ROS release are indirectly dependent on the radiant energy rather than the radiant power (8). In 2014, R Salman Yazdi et al. continuously irradiated the sperm of patients with asthenospermia with a wavelength of 830 nm at different radiant energies. Their results showed that sperm were able to move at 4 J/cm2 and 6 J/cm2 (44). Therefore, for a more effective laser treatment, it is necessary to check the appropriate energy dose. In this study, three low-energy doses were used, all three of which showed significant results by examining the degree of cell differentiation and sperm fertility. However, the effect of energy doses of 2 J/cm2 and 4 J/cm2 were the same and there was no significant difference between these two energy doses. On the other hand, measuring SI, which indicates sperm production, was almost equally effective in all three groups. In general, the results of our research show that reducing the dose of energy has a better effect on improving the process of spermatogenesis and treatment of azoospermia.
In a similar study to ours, Busulfan-induced infertile rats were treated with an energy density of 0.03 and 0.2 J/cm2. In this study, a pulsed infrared laser with a wavelength of 890nm was used. Examination of sperm parameters, measurement of serum testosterone level, TUNEL assay, and histomorphological evaluation showed that a lower energy density of 0.03 J/cm2 caused a significant increase in the number of germ cells and Leydig cells and decreased apoptosis (40). This shows that in addition to radiation energy, a parameter such as the radiated wavelength used is also effective in terms of the effectiveness of laser treatment. In the present study, which used a diode laser with a wavelength of 808 nm, a higher energy dose of 8 J/cm2 showed better results than the other two groups. For example, it improved testicular tissue repair faster than the other two groups and the diameter and thickness of the epithelium of the seminal vesicles and differentiated cell layers were better visible. The majority of studies, together with the results of our study, confirm that low-energy laser radiation has the best effect on activating cellular mechanisms and improving metabolism, and this non-invasive method is the best option in the treatment of infertility-related diseases such as azoospermia.
To use this non-invasive and safe in-vivo treatment on the human testicle requires further research and evaluation of the reproducibility of these findings in vitro as well as measuring the harmful effects on DNA integrity.
Conclusion
In this investigation, the employment of a laser with an energy density of 8 J/cm2 was shown to be beneficial in boosting germ cell and sperm production. Although the two groups of 2 J/cm2 and 4 J/cm2 had positive effects on sperm production, the discrepancies were not statistically significant in production.
Authors’ Contributions
Conceptualization, designing project, data curation: H.T, M.M and E.S… Project administration, Material preparation, Data collection: H.T, A.S, F.F, Z.A… Statistical analysis: R.Sh… writing original draft: Z.A and F.F… writing-review-editing: all authors
Conflict of Interest
None.
Acknowledgement
The authors acknowledge the Biophotonic and Biotechnology Research Center of Azad University of Tabriz for its scientific support.
Funding
This project has been funded by the Islamic Azad University of Tabriz
Consent to Participate
Yes
Consent for Publication
Yes
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