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Oocyte cryopreservation is an essential procedure in assisted reproductive technologies, aimed at preserving fertility, particularly for women undergoing IVF treatment or at risk of ovarian damage due to radiation, chemotherapy, or surgery. Despite its growing use, the survival and fertilization rates of cryopreserved oocytes remain suboptimal, largely due to cryo-induced oxidative stress. The generation of Reactive Oxygen Species (ROS) during freezing and thawing causes considerable damage to key cellular components, including proteins, lipids, DNA, and mitochondria. This oxidative stress compromises oocyte quality and reduces developmental potential. To address these challenges, the use of additives - especially antioxidants - has shown significant promise in mitigating oxidative damage. Enzymatic antioxidants such as Superoxide Dismutase (SOD) and Catalase (CAT), along with non-enzymatic antioxidants like glutathione, melatonin, and resveratrol, have demonstrated the ability to neutralize ROS and improve oocyte viability and developmental outcomes. Recent studies highlight the potential of Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to effectively counteract mitochondrial ROS and enhance cellular defense mechanisms during cryopreservation. This review explores the cellular mechanisms of cryodamage, the role of oxidative stress in oocyte cryopreservation, and the potential of various antioxidant strategies to enhance oocyte survival and function. Developing effective antioxidant supplementation approaches may significantly improve the outcomes of cryopreservation in reproductive medicine.

Main objective Due to Human Wharton’s Jelly (HWJ) could be applied in tissue engineering as a bio scaffold, the present study was conducted to investigate the effects of HWJ hydrogel on in vitro culture and auto-transplantation of mouse ovarian follicles. Materials and methods HWJ was isolated from umbilical cord and decellularized with SDS/Tris/EDTA. DNA, Collagen and Glycosaminoglycans (GAGs) were measured. Decellularized Wharton’s Jelly (DWJ) was dissolved to make Wharton’s Jelly Hydrogel (WJH), and composited with Alginate (ALG) (1.5%) in equal ratio (WJH+ALG). Then, mouse preantral follicles were isolated and encapsulated in 10μL droplets of WJH and randomly considered for both 14 days culture and auto-transplantation. Results Collagen, GAGs and DNA evaluations showed majority of WJ cells have been removed and MTT approved no toxicity. Degradation rate and rheological analysis represented optimal hydrogel compatibility. The data from in vitro culture revealed significant antral formation in WJH+ALG (P≤0.05). In transplantation, follicles failed to survive in ALG; however, survived in WJH+ALG to antral stage (P<0.05). VEGF and CD34 had greater expression in WJH+ALG than ALG (P< 0.05). Conclusion Wharton’s jelly hydrogel and Alginate compound is interesting composite for successful development of mouse preantral follicles in both 3D in vitro culture and transplantation.

Objective: In vitro maturation (IVM) and cryopreservation of oocytes are two important parts of assisted reproductive technology (ART), but their efficacy is low. This study aimed to improve the quality of in vitro vitrified-warmed maturated oocytes using granulosa cell conditioned medium (GCCM). Materials and Methods: In the experimental study, fresh/non-vitrified and vitrified-warmed mouse germinal vesicle (GV) oocytes (as F and V) were in vitro maturated using basal medium (BM) and also BM supplemented with 50% GCCM as treated groups (GM), and categorized as FBM, FGM, VBM and VGM groups, respectively. The rate of successful IVM (MII oocyte formation), mitochondrial membrane potential and the viability of MII oocytes were determined using inverted microscopy, JC-1 and trypan blue staining. Then, the rate of in vitro fertilization (IVF) and subsequent two-cell embryo formation was calculated. Finally, the expression levels of Oct4, Sox2, Cdk-2, Gdf9, Integrin beta1 and Igf2 were analyzed using real-time polymerase chain reaction (PCR) in MII oocytes and two-cell embryos. Results: These analyses showed that GCCM significantly increased the IVM rate, oocyte meiotic resumption and mitochondrial membrane potential (P<0.05). In addition, the rate of IVF and two-cell embryo formation was significantly higher in FGM and VGM compared to FBM and VBM (P<0.05). Interestingly, GCCM significantly affected the expression of the studied genes. Conclusion:Our findings suggest that GCCM might be useful for improving the efficiency of IVM and the subsequent IVF outcomes.

The high miscarriage rates that result following transfer of embryos derived from in vitro maturation (IVM) of oocytes necessitate improvements in the processes involved. This study aimed to improve the quality of in vitro matured oocytes using granulosa cell conditioned medium (GCCM) as the culture medium. In this work, germinal vesicle (GV)-stage oocytes from NMRI mice were collected and cultured using three types of culture medium: Base medium (BM) (control), 50% granulosa cell conditioned medium (GCCM50) and 100% GCCM (GCCM100). After IVM, the mitochondria activity potential and viability of metaphase II (MII) oocytes were evaluated by JC-1 and trypan blue staining, respectively. Maturational gene expression levels of CyclinB1, Cdk1 and Gdf9 in the control, GCCM50 and GCCM100 samples were analyzed using real-time polymerase chain reaction (PCR). The viability rate of in vitro matured oocytes was highest in the GCCM50 group. JC-1 staining showed that GCCM50 enhances mitochondrial activity more than the other groups (P < 0.05). Gene expression levels of Cdk1 and Gdf9 were higher in the group with GCCM50 treatment, than in the control and GCCM100 groups (P < 0.05), while the expression level of CyclinB1 did not differ among the groups. The results indicated that a 50% concentration of GCCM in combination with BM components enhanced MII and viability rates and mitochondria activity of mouse immature oocytes.

Main objectiveDue to Human Wharton's Jelly (HWJ) could be applied in tissue engineering as a bio scaffold, the present study was conducted to investigate the effects of HWJ hydrogel on in vitro culture and auto-transplantation of mouse ovarian follicles.Materials and methodsHWJ was isolated from umbilical cord and decellularized with SDS/Tris/EDTA. DNA, Collagen and Glycosaminoglycans (GAGs) were measured. Decellularized Wharton's Jelly (DWJ) was dissolved to make Wharton's Jelly Hydrogel (WJH), and composited with Alginate (ALG) (1.5%) in equal ratio (WJH+ALG). Then, mouse preantral follicles were isolated and encapsulated in 10μL droplets of WJH and randomly considered for both 14 days culture and auto-transplantation.ResultsCollagen, GAGs and DNA evaluations showed majority of WJ cells have been removed and MTT approved no toxicity. Degradation rate and rheological analysis represented optimal hydrogel compatibility. The data from in vitro culture revealed significant antral formation in WJH+ALG (P≤0.05). In transplantation, follicles failed to survive in ALG; however, survived in WJH+ALG to antral stage (P<0.05). VEGF and CD34 had greater expression in WJH+ALG than ALG (P< 0.05).ConclusionWharton's jelly hydrogel and Alginate compound is interesting composite for successful development of mouse preantral follicles in both 3D in vitro culture and transplantation.