In Vitro Development of Mouse Preantral Follicle with Using Amniotic Membrane Extract-Loaded Hydrogels.

An artificial ovary based on the alginate (ALG) hydrogel has been widely implemented to preserve prepubertal female fertility. However, this platform is not fully capable of successful an ovary microenvironment simulation for follicle development, holding great potential for its improvement. Therefore, this experimental study aimed to evaluate the effect of 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 adjusted for the follicles encapsulated in the ALG-AME hydrogel. Results demonstrated that 100 mIU/ml FSH showed a significant follicle survival rate compared with 10 mIU/ml FSH (P=0.005). According to MTT assay finding, the rate of weight loss, and rheology evaluations, ALG containing 1 mg/ml AME was identified as an optimal sample of follicle culture instead of other AME concentrations. Follicle diameter significantly increased in the ALG-AME 1 hydrogel compared with the ALG control group without AME (P=0.027). The storage modulus of ALG-AME 1 was 773 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 spindle rate in comparison with the control group. It can be concluded that ALG-AME 1 could not significantly impact the mouse preantral follicle.

Cryopreservation of Limited Sperm Using A Combination of Sucrose and Taurine, Loaded on Two Different Devices, and Thawed at Two Different Temperatures.

BACKGROUND: Cryopreservation of sperm is essential for patients with low sperm counts and couples undergoing infertility treatment. The aim of this study was to compare the effects of Taurine (T) and Sucrose (S) in individual sperm cryopreservation utilizing cryotop and petri dish and thawing at 37 and 42°C. MATERIALS AND METHODS: In this experimental study, 17 normospermic semen samples were processed using the "Swim-up" procedure and progressively motile sperm were then isolated from these samples using an inverted microscope. Sperm were added to droplets of "sucrose medium" with 25 mM Taurine antioxidant (S+T) and the commercial cryoprotectant "Sperm Freeze" (CPA), loaded on a petri dish and cryotop. After rapid freezing of the samples, they were thawed at two different temperatures (37°C and 42°C), and the sperm classical parameters, viability, and DNA fragmentation were assessed. RESULTS: Statistical analysis displayed a significant increase in total and progressive motility in individual sperm freezing on cryotop with CPA and thawing at 42°C (P<0.05). Other parameters did not show any differences between the CPA and S+T groups and two thawing temperatures in either of the cryopreservation methods. CONCLUSION: Although, both cryoprotectants (CPA and S+T) may preserve individual sperm effectively using cryotop, the CPA and thawing at 42°C showed a better effect on the motility percentage of the small number of sperm.

In-vitro generation of follicle-like structures from human germ cell-like cells derived from theca stem cell combined with ovarian somatic cells.

BACKGROUND: The objective of this study was to induce the differentiation of human theca stem cells (hTSCs) into germ cell-like cells (hGCLCs) and assess their developmental progression following in vitro 3D culture with ovarian somatic cells within the follicle-like structures. To achieve this, the hTSCs were isolated from small antral follicles of three patients of varying ages and were then seeded in a differentiation medium for 40 days. The differentiated hGCLCs were subsequently aggregated with somatic ovarian cells (cumulus cells and hTSCs) in a ratio of 1:10 and cultured in a growth medium in a suspension culture dish. In addition to examining the morphologies, sizes, and viabilities of the differentiated hGCLCs, this study also analyzed the expression of DAZL and GDF9 proteins within the follicle-like structures. RESULTS: After 12 days, the hTSCs began to differentiate into hGCLCs, with their shapes changing from spindle-shaped to spherical. The sizes of hGCLCs increased during the differentiation period (from 25 µm to 50 µm). The survival rate of the hGCLCs after differentiation and in vitro development in primordial follicle-like structures was 54%. Unlike hTSCs, which did not express the DAZL protein, the hGCLCs and follicle-like structures successfully expressed DAZL protein (P-value < 0.05). However, hGCLCs poorly expressed the GDF9 protein. Further, the culture of hGCLCs in primordial follicle-like structures significantly increased GDF9 expression (P-value < 0.05). CONCLUSION: In conclusion, our study demonstrated that 3D cultures with ovarian somatic cells in follicle-like structures caused the successful differentiation of reproducible hGCLCs from hTSCs derived from three patients of different ages. Moreover, this method not only enhanced the in vitro development of hGCLCs but also presented a novel approach for co-culturing and developing in vitro oocyte like cells, ultimately leading to the production of artificial follicles.