The healing effects of platelet rich plasma and exosomes derived from stem cells on the spermatogenic proteins and sperm parameters of non-obstructive azoospermia model rats
The healing effects of platelet rich plasma and exosomes derived from stem cells on the spermatogenic proteins and sperm parameters of non-obstructive azoospermia model rats
Hoda Fazaeli,1Azar Sheikholeslami,2,*Faezeh Davoodi Asl,3Mohsen Sheykhhasan,4Leila Naserpour,5Naser Kalhor,6
1. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture and Research, Qom, Iran 2. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture and Research, Qom, Iran 3. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture and Research, Qom, Iran 4. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture and Research, Qom, Iran 5. Department of Reproductive Biology, Academic Center for Education, Culture and Research, Qom Branch, Qom, Iran. 6. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture and Research, Qom, Iran
Introduction: Azoospermia with two types of obstructive (OA) and non-obstructive (NOA) accounts for a significant percentage of male infertility. Patients with NOA are unable to have children of their own, and they usually have to resort to adoption or the use of donated sperm. Due to the irreversible nature of spermatogenesis damage in patients with NOA, testicular biopsy and assisted reproductive techniques are the only ways to obtain biological offspring. However, these methods have limited success and do not work for all cases. Therefore, the current challenge is to improve the spermatogenic function of azoospermic men to generate sperm in their ejaculate or to improve the probability of successful sperm retrieval from the testis for ICSI. In this way, researchers are recently trying to develop treatments based on stem cell transplantation for azoospermia. It has been shown that extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are the main agents responsible for exerting the paracrine effects and consequently biological functions of MSCs, which offer significant advantages in the field of regenerative medicine compared to their cells of origin. EVs can bypass most of the safety considerations associated with direct cell transplantation, and unlike transplanted cells that cannot be recovered, treatment using EVs is not permanent and can be easily stopped if adverse effects occur. An autologous concentration of human platelets in a small amount of plasma is known as platelet-rich plasma (PRP). The generation of reproductive hormones and improvement of spermatogenesis in patients with poor sperm parameters, as well as testicular torsion are both enhanced by the platelet-rich plasma. In this study, comparative effect of exosomes obtained from MSCs derived from adipose tissue (AD-Exo) and PRP was evaluated on the recovery of spermatogenesis in non-obstructive azoospermia model rats.
Methods: We examined the effects of human PRP and exosomes generated by adipose tissue-derived MSCs (AD-MSCs) on the restoration of spermatogenesis after intra-testis injection in NOA rat models, which was carried out on 30 male 8–12 weeks old Wistar rats classified in 5 groups. AD-MSCs were obtained and grown up to passage 3 in the cell culture facility. The exosomes were then isolated from conditioned medium of the cells. In addition, we collected peripheral blood from willing donors and PRP was separated. Rats were given two intraperitoneal injections of busulfan (10 mg/kg body weight) with 21 days interval in order to generate NOA model. Intratesticular injection of 100 microliters of exosomes (500 mg/mL), 100 microliters of PRP, and 100 microliters of PBS was done in the AD-Exo, PRP, and sham groups, respectively, at the time points of three days and two weeks after induction. The NOA-induced rats with no treatment were considered as the positive-control (NOA) group and the healthy rats with no induction and no treatment were supposed as negative-control group. Two months after final medication, the rats were put down for further research. In order to investigate the changes in different groups of NOA model rats, western blot analysis was done for DDX4 and DAZL protein. In addition, the amount of count and motility in the rat sperm samples of different groups were evaluated. Finally, the resulting data were statistically analyzed using SPSS software.
Results: Based on the analysis of the experimental groups, it was discovered that the expression of the DAZL and DDX4 was significantly higher in the AD-Exo and PRP treatment groups than in the control group, while it was significantly decreased in the NOA and sham groups (p≤ 0.05). The DAZL and DDX4 protein appears to be restored as a result of exosome and PRP treatment. In addition, compared to the control group, sperm parameters were considerably greater in the AD-Exo and PRP treatment groups, while they were substantially reduced in the NOA and sham groups (p≤ 0.05).
Conclusion: This investigation verified the beneficial effects of PRP and exosomes released by AD-MSCs on the spermatogenesis-related proteins and sperm parameters. To ascertain the therapeutic benefit of these medications on patients with NOA, it is evident that more research on testicular histology, size and weight of testis is needed.