Extracellular matrix-derived scaffolds provide an efficient platform for the generation of organ-like structures. Successful development of testicular organoids (TOs) with the capability of supporting complete spermatogenesis has not been reported yet. Here, we have developed an optimized method for the decellularization of ram testicular tissue fragments. Our findings showed that testicular fragments treated with a serial combination of Triton X-100 and SDS in PBS for 48 h resulted in the efficient removal of cellular materials and retention of the extracellular matrix (ECM) components. In order to fabricate testis-derived scaffolds (TDSs), the testicular ECM (T-ECM) was digested in acid/pepsin, followed by neutralization of pre-gel solution to form a hydrogel. Then, the hydrogels were freeze-dried and cross-linked using a chemical method. To reach the optimal concentration for the T-ECM in the fabrication of TDSs, the scaffold properties including porosity, pore size, swelling behavior, and degradation were evaluated. Our study suggested that 25 mg ml⁻¹ of the T-ECM is the best concentration for the fabrication of macroporous TDSs for demonstrating lower pore size, homogeneously distributed pores, and a higher swelling ratio. Furthermore, inoculation of neonatal mouse testicular cells onto TDSs resulted in the generation of multicellular TOs in which the differentiation of spermatogonial cells into post-meiotic cells was confirmed. Hormonal analysis of TDSs revealed the functionality of TOs in the secretion of testosterone and inhibin B. The current study also demonstrated that macroporous TDSs could provide a novel platform for testicular tissue engineering and in vitro spermatogenesis.

The purpose of this study was to evaluate the effects of cryotop vitrification of sheep cumulus-oocyte complexes (COCs) on oocyte maturation, apoptotic gene expression and incidence of chromosomal abnormalities. Freshly isolated (control group) and vitrified-warmed COCs (cryotop group) were matured in vitro. The expression of pro- and anti-apoptotic genes was investigated by real-time PCR. The incidence of numerical chromosomal abnormalities was evaluated by cytogenetic analysis. The mean percentage of oocytes in the cryotop and control groups that reached metaphase II was 49.25 ± 3.01% and 51.94 ± 2.7% respectively. The expression rates of pro- and anti-apoptotic genes were similar in both groups, whereas the incidence of numerical chromosomal abnormalities was higher in the cryotop group compared to the control group (42.5% vs. 20%, p < 0.05). Although cryotop vitrification of COCs did not affect the incidence of oocyte maturation or apoptotic gene expression, significant deficiencies in the maintenance of oocyte chromosomal organization were seen.

Large-scale production and noninvasive methods for harvesting mesenchymal stem cells (MSCs), particularly in elderly individuals, has prompted researchers to find new patient-specific sources for MSCs in regenerative medicine. This study aims to produce MSCs from human induced pluripotent stem cells (hiPSCs) and to evaluate their therapeutic effects in a CCl₄-induced mouse model of fulminant hepatic failure (FHF). hiPSC-MSCs have shown MSC morphology, antigen profile and differentiation capabilities, and improved hepatic function in our model. hiPSC-MSC-transplanted animals provide significant benefit in terms of survival, serum LDH, total bilirubin, and lipid peroxidation. hiPSC-MSC therapy resulted in a one-third reduction of histologic activity index and a threefold increase in the number of proliferating hepatocytes. This was accompanied by a significant decrease in the expression levels of collagen type I, Mmp13, Mmp2, and Mmp9 genes and increase in Timp1 and Timp2 genes in transplanted groups. hiPSC-MSCs secreted hepatocyte growth factor (HGF) in vitro and also expressed HGF in evaluated liver sections. Similar results were observed with human bone marrow (hBM)-derived MSCs. In conclusion, our results have demonstrated that hiPSC-MSCs might be valuable appropriate alternatives for hBM-MSCs in FHF liver repair and support liver function by cell therapy with a large-scale production capacity, patient-specific nature, and no invasive MSC harvesting.

This study evaluates the relationship between sperm intracellular reactive oxygen species (ROS; H2 O2 , O2 ), DNA fragmentation (DF), low mitochondria membrane potential (MMP) of sperm and normal pronuclear formation among intracytoplasmic sperm injection (ICSI) patients. Semen samples were obtained from 62 infertile male who were candidates for ICSI treatment. After sperm processing, metaphase II (MII) oocytes were injected, and the mean percentages of intracellular ROS, MMP and DF were evaluated using flow cytometry. The mean percentages of pronuclear formation and zygote score (Z) were also recorded, and Pearson, Spearman's rank correlation coefficient and Kruskal-Wallis tests were applied to analyse the data. The amounts of sperm intracellular H2 O2 and O2-˙ had significant positive correlation with low MMP (P < 0.01). The intracellular ROS had a negative correlation with pronuclear formation (P < 0.05), and its effect was higher than 66.66%. In addition, the mean percentages of neither H2 O2 nor O2-˙ affected the quality of pronuclear embryos (Z-score). This study shows that although high levels of both sperm intracellular H2 O2 and O2-˙ in ICSI patients have deleterious effect on sperm MMP, only H2 O2 may interfere in pronuclear formation.