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Background: The utilization of decellularized extracellular matrix (dECM) derived from animal testis tissue hasdemonstrated potential as a component of tissue-specific scaffolds. Current research is mostly centered arounddECM as a natural resource for culturing testicular cells. This study aimed to assess firstly the comparison ofVoytik-Harbin (VH) and Frytes protocol in creating Ram’s dECM testis hydrogel and secondly the evaluation ofthe best protocol effect on in vitro spermatogenesis.Materials and Methods: In this experimental study, the six testes of mature rams were decellularized and the hydrogelproduction was performed by i. The Frytes protocol utilized a concentration of 1 mg/mL of pepsin, dissolvedin either 0.1 or 0.01 M HCl, and ii. The VH protocol was involved 10 mg of pepsin per 100 mg of ECM in 0.5 M ofacetic acid. Subsequently, mouse testicular cells were cultivated on collagen hydrogel as the control and the moreeffective testicular-derived hydrogel (TDH) to evaluate the early stages of in vitro spermatogenesis.Results: While the Freytes protocol produced a homogeneous pre-gel solution with both HCl concentrations; elevatingthe pH to 7.4 loosened the hydrogel and made gelation problematic. In contrast, the VH protocol solidifiedthe hydrogel and produced a strong hydrogel due to its gelation consistency. Furthermore, the prepared hydrogel byVH with 25 mg of dECM had a significantly higher priority in terms of rheology and structure (P more...

An artificial ovary based on the alginate (ALG) hydrogel has been widely implemented to preserve prepubertal femalefertility. However, this platform is not fully capable of successful an ovary microenvironment simulation for follicledevelopment, holding great potential for its improvement. Therefore, this experimental study aimed to evaluate the effectof an amniotic membrane extract (AME) -loaded hydrogel on the mouse preantral follicles in vitro development.In order to have better follicle development, first, the impact of different concentrations of follicle-stimulating hormone(FSH) was evaluated on the mouse preantral follicles encapsulated in ALG. Later, the appropriate dose was adjustedfor the follicles encapsulated in the ALG-AME hydrogel. Results demonstrated that 100 mIU/ml FSH showeda significant follicle survival rate compared with 10 mIU/ml FSH (P=0.005). According to MTT assay finding, therate of weight loss, and rheology evaluations, ALG containing 1 mg/ml AME was identified as an optimal sampleof follicle culture instead of other AME concentrations. Follicle diameter significantly increased in the ALG-AME 1hydrogel compared with the ALG control group without AME (P=0.027). The storage modulus of ALG-AME 1 was773 Pa and retained the follicle morphology for 13 days. No statistically substantial difference was seen in survival,antrum cavity formation, and competent oocyte in terms of the normal chromosomal arrangement and meiotic spindlerate in comparison with the control group. It can be concluded that ALG-AME 1 could not significantly impact themouse preantral follicle. more...

Objective In the present study, the applicability of hyaluronic acid-alginate (HAA) hydrogel and ovarian cells (OCs) for the culture of mouse ovarian follicles were investigated and compared with those of alginate (ALG) and fibrin-alginate (FA) hydrogels. Materials And Methods In the first step of this experimental study, mechanically isolated preantral follicles from the ovaries of two-week-old mice were encapsulated in the absence or presence of OCs in ALG, HAA, and FA hydrogels and cultured for 14 days. The morphology, diameter, survival and antrum formation rates of the follicles and the maturation and quality of the oocytes were evaluated during culture. In the second step, preantral follicles were cultured similar to the first step, but for 13 days, and their gene expressions and hormonal secretion were assessed on the last day of culture. Results In the absence of OCs, higher numbers of ALG- and HAA-encapsulated follicles reached the antral stage compared to FA-encapsulated follicles (P more...

Background In the present study, the effects of alginate (ALG) concentration and ovarian cells (OCs) on the devel- opment and function of follicles were simultaneously evaluated. Materials and Methods In the first step of this experimental study, preantral follicles were isolated from the ovaries of 2-week-old mice, encapsulated in the absence or presence of OCs in 0.5, 0.75 and 1% ALG hydrogels, and cultured for 14 days. The morphology, diameter, survival and antrum formation rates of the follicles and the maturation of the oocytes were evaluated during culture. In the second step, preantral follicles were cultured in the best chosen ALG concentration, in both the absence and presence of OCs. Following these steps, the amount of DNA fragmentation, the expression levels of connexin 37 and connexin 43 proteins, the secretion levels of estradiol, progesterone and andros- tenedione by the follicles and the quality of mature (MII) oocytes were assessed. Results Our data revealed that in the absence of OCs, follicles of 0.5% group showed a higher survival rate than the 0.75 and 1% groups (71.87 vs. 52.52 and 40%, respectively, P < 0.05). Nonetheless, the antrum formation rate of the 1% group was higher and its oocyte degeneration rate was lower than that in the other groups. Furthermore, it was observed that co-culture of follicles with OCs relatively increased the follicle diameter, survival, antrum formation, and germinal vesicle (GV) to GV break down (GVBD)/MII transition rates. At last, the comparison of 0.5%-OCs and 0.5%+OCs groups indicated that the co-culture condition resulted in more progesterone production (1.8 ± 0.2 vs. 3.2 ± 0.4 ng/ml, respectively, P < 0.05) and also decreased oocytes’ cortical granule abnormalities (100 vs. 40% for 0.5%- OCs and 0.5%+OCs groups, respectively). Conclusion The present study revealed that 0.5% ALG hydrogel is relatively suitable for preantral follicle culture, and in the presence of OCs, it mimics the natural ovarian condition better than the higher concentrations of ALG hydrogel. more...