Successful 3D culture and transplantation of mouse isolated preantral follicles in hydrogel of bioengineered Wharton's jelly.

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.

Granulosa Cell Conditioned Medium Enhances The Rate of Mouse Oocyte In Vitro Maturation and Embryo Formation.

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.

Mouse ovarian follicle growth in an amniotic membrane-based hydrogel.

Three-dimensional cultures of follicles on ECM-based scaffolds can be an approach for women who become infertile after cancer treatments. Human amniotic membrane (HAM) is extensively employed in tissue engineering because of its unique properties. We cultured mouse pre-antral follicles in a hydrogel derived from decellularized amniotic membrane (DAM) combined with alginate (ALG) to improve ovarian follicle culture. HAM was decellularized. Quantitative (nuclear contents, collagen, glycosaminoglycan [GAG]) and qualitative (DAPI, H&E, Masson's trichrome, Alcian blue, scanning electron microscopy assessments were performed. Then, we created an amniotic membrane-based hydrogel (AMBH) and conducted AMBH characterization assays (rheology, MTS, degradation rate). Isolated mouse pre-antral follicles were cultured in 15 mg/mL AMBH (AMBH15), 30 mg/mL AMBH (AMBH30), or 45 mg/mL AMBH (AMBH45). ALG hydrogel was the control group. Follicular diameters, estradiol hormone secretion rate, follicular morphology, and the follicle antral and degeneration rate were examined. Quantitative and qualitative assays indicated successful decellularization. AMBH characterization assays showed that the ALG hydrogel had more appropriate gelation and slower degradation than AMBH. There was a statistically higher antral follicle formation rate in the AMBH45 group (p < .05) compared to the AMBH30 and AMBH15 groups and less (p < .05) degenerated follicles. There was no significant difference with the ALG group. Diameter and estradiol hormone secretion in the AMBH45 group were not significantly higher than the ALG group. Although decellularization was confirmed and the viscoelastic parameters of AMBH support follicle culture, there was no significant effect on ovarian follicle maturation compared to the ALG control group.

Survey of Third-Party Parenting Options Associated With Fertility Preservation Available to Patients With Cancer Around the Globe.

Purpose: In the accompanying article, "Survey of Fertility Preservation Options Available to Patients With Cancer Around the Globe," we showed that specific fertility preservation services may not be offered at various sites around the world because of cultural and legal barriers. We assessed global and regional experiences as well as the legal status of third-party reproduction and adoption to serve as a comprehensive international data set and resource for groups that wish to begin oncofertility interventions. Methods: We provide data on the legalities of third-party assisted reproductive technologies and other family-building options in the 28 oncofertility-practicing countries surveyed. Results: We found regional and country differences that will be important in the development of tailored resources for physicians and for patient brochures that are sensitive to these local restrictions and cultural norms. Conclusion: Because many patients first consult Web-based materials, the formal assessment of the availability of these options provides members of the global oncofertility community with data to which they might otherwise not have ready access to better serve their patients.

Survey of Fertility Preservation Options Available to Patients With Cancer Around the Globe.

Purpose: Oncofertility focuses on providing fertility and endocrine-sparing options to patients who undergo life-preserving but gonadotoxic cancer treatment. The resources needed to meet patient demand often are fragmented along disciplinary lines. We quantify assets and gaps in oncofertility care on a global scale. Methods: Survey-based questionnaires were provided to 191 members of the Oncofertility Consortium Global Partners Network, a National Institutes of Health-funded organization. Responses were analyzed to measure trends and regional subtleties about patient oncofertility experiences and to analyze barriers to care at sites that provide oncofertility services. Results: Sixty-three responses were received (response rate, 25%), and 40 were analyzed from oncofertility centers in 28 countries. Thirty of 40 survey results (75%) showed that formal referral processes and psychological care are provided to patients at the majority of sites. Fourteen of 23 respondents (61%) stated that some fertility preservation services are not offered because of cultural and legal barriers. The growth of oncofertility and its capacity to improve the lives of cancer survivors around the globe relies on concentrated efforts to increase awareness, promote collaboration, share best practices, and advocate for research funding. Conclusion: This survey reveals global and regional successes and challenges and provides insight into what is needed to advance the field and make the discussion of fertility preservation and endocrine health a standard component of the cancer treatment plan. As the field of oncofertility continues to develop around the globe, regular assessment of both international and regional barriers to quality care must continue to guide process improvements.

Maturational gene upregulation and mitochondrial activity enhancement in mouse in vitro matured oocytes and using granulosa cell conditioned medium.

SummaryThe 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.