mahya ramesh

Testicular ischemia/reperfusion injury, a significant result of testicular torsion, can lead to the risk of male infertility.

The current study aimed to evaluate the effect of human amniotic membrane-derived mesenchymal stem cells (hAMSCs) secretome on testicular torsion/detorsion (T/D) in mice.

All the experiments were performed in the Anatomy Department of Tehran University of Medical Sciences, Tehran, Iran, during the period of March 2023 to December 2023. 40 male NMRI mice (5-7 wk, 25-30 gr) were randomized into: 1) the sham group: mice received sham operations with no other interventions, 2) T/D group, 3) negative control group; torsion detorsion + intratesticular injection of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, and 4) the T/D group + hAMSCs secreted factors. Serum testosterone levels, hematoxylin and eosin staining, and sperm quality parameters were used to evaluate the therapeutic effects of hAMSCs secreted factors on the testicular structure and function. Tissue oxidative stress was measured by determining malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase-1. Nuclear factor erythroid 2-related factor 2, Kelch-like ECH-associated protein 1, NAD-dependent deacetylase sirtuin-1, tumor necrosis factor-alpha and tumor protein P53 mRNA expressions were assessed in testis via real-time polymerase chain reaction.

The results showed that hAMSCs secreted factors alleviated testicular T/D injury by attenuating oxidative stress, inflammatory response, and apoptosis via modulating the sirtuin-1/ nuclear factor erythroid 2-related factor 2/tumor necrosis factor-alpha signaling pathway.

hAMSCs secreted factors increased antioxidative, anti-inflammatory, and antiapoptotic properties which consequently increased testosterone levels, spermatogenesis, and sperm quality parameters.

Testicular torsion leads the a disorder of contralateral testicular function in addition to destructive effects on the ipsilateral testicle through the impairment of the blood- testis barrier, release of sperm into the blood, activation of immune responses, and production of anti-sperm antibody. Detorsion surgery also resulted in inflammatory reactions and the production of reactive oxygen species (ROS), which was finally accompanied by the destruction of the contralateral testicular tissue and infertility. The application of mesenchymal stem cells conditioned medium recently has been considered in the treatment of male infertility due to the presence of various growth factors and anti-inflammatory properties. The aim of this study was to evaluate the effect of conditioned medium derived from human amniotic membrane mesenchymal stem cells on the promotion of spermatogenesis and sperm chromatin structure in the contralateral testis following the torsion of the unilateral spermatic cord.

In this experimental study 40 adult male NMRI mice were randomly divided into 4 groups. Control group without surgical intervention. Left spermatic cord torsion / detorsion group. Negative control group: torsion / detorsion surgery and injection of culture medium into the left testis. Treatment group: torsion / detorsion surgery and injection of conditioned medium derived from human amniotic membrane mesenchymal stem cells into the left testis. The Histomorphometric evaluation of the right testicle was done by preparation of tissue sections and hematoxylin and eosin (H&E) staining. This evaluation included counting different cells of testicular tissue, the number of seminiferous tubules per field, measurement of outer diameter and epithelium thickness of tubules, Johnson's score, and tubules spermatogenic criteria by Image J software. Also, semen samples were prepared from the cauda epididymis and toluidine blue staining was performed for evaluation of sperm chromatin integrity. One- way analysis of variance (ANOVA) and Tukey's test were performed for comparisons between groups, p-value less than 0.05 (P ≤ 0.05) is statistically significant.

The number of spermatogonial cells, primary spermatocyte, spermatid, Sertoli, Leydig, Johnson's score, outer diameter and thickness of seminiferous tubules germinal epithelium, TDI, SPI, MI, and the diameter of the spermatogonial cell nucleus in right testis significantly decreased in the torsion / detorsion group (P < 0.01). A remarkable improvement in the above parameters was observed after treatment with the conditioned medium in comparison with the torsion / detorsion group, although there was a significant difference as compared to the control group (P < 0.01). The percentage of sperms with abnormal chromatin in the spermatic cord torsion / detorsion group increased significantly compared to the control group (19.6 ± 8.54) (P < 0.001). A significant improvement in the sperm chromatin structure was observed compared to the torsion/ detorsion group after treatment (14.4± 3.6) (P< 0.05).

It seems that Conditioned medium derived from human amniotic membrane mesenchymal stem cells leadsto a relative improvement in sperm chromatin and spermatogenic indices of the contralateral testicular tissue after unilateral ischemia / reperfusion injury of the spermatic cord.

Torsion of spermatic cord induces ischemic-reperfusion injury which leads to testicular dysfunction, apoptosis of germ cells, ROS generation and infertility. Platelet-rich plasma (PRP) is a rich source of growth factors and anti-inflammatory cytokines. In this study, the effects of PRP on spermatogenesis and sperm chromatin condensation after ischemia-reperfusion injury were evaluated.

Forty adult male mice were procured in the study. Control group, torsion/ detorsion group, negative control group: torsion/ detorsion surgery with intra testicular injection of culture medium, treatment group: torsion/ detorsion surgery with intra testicular injection of PRP. After one cycle of spermatogenesis, histomorphometric evaluation was performed by PAS staining and sperm chromatin condensation was assessed by aniline blue staining. Data was analyzed by using SPSS Statistics and ANOVA followed by Tukey test.

The mean number of spermatogenic cells, Sertoli, Leydig, Myoid, Johnson score and mean diameter and thickness of germinal epithelium were significantly decreased following ischemia-reperfusion injury. Mean thickness of seminiferous tubules basement membrane as well as sperm percentage with residual histone increased after ischemia-reperfusion injury (P <0.001). After PRP treatment, there was a significant improvement in the histological structure of the tubules, cell viability, and sperm chromatin condensation (P <0.001). While, a significant difference was observed in comparison with control group.

PRP can partially ameliorate the complications of ischemia-reperfusion injury through its growth factors and anti-inflammatory cytokines involved in angiogenesis and antioxidant activity.