Dados do Trabalho

Title

Chemically defined 3D matrix for in vitro maturation (IVM) of human oocytes

Objective

We reported earlier the use of a nontoxic PVA-based hydrogel modified with BTCA, for fibroblasts and stem cells culture. The objective of the present study is to test the use of a PVA-based/BTCA gel for the culture of human immature oocytes to improve in vitro conditions that may enhance present oocyte maturation rates. Also, there is the need to find easily manufactured, safe, and economical materials for human IVM, particularly for fertility preservation in cancer patients.

Methods

Immature human oocytes and cumulus cells were obtained with informed consent from patients undergoing conventional IVF cycles after superovulation and hCG priming. Cumulus cells and oocytes were cultured in CSC medium supplemented with 10% SSS in a 10% PVA (Mw 130,000, 99% degree of hydrolysis, Sigma Chemical Co., St. Louis, MO) and BTCA (1,2,3,4-butanetetracarboxylic acid, Sigma Chemical Co., St. Louis, MO, 99%) matrix for 24hrs.
PVA was added to 30 mL deionized water at 100o.C. After dissolution, 0.3g of BTCA was added and stirred for 20 minutes at 60o.C.The solution was placed in polystyrene Petri dishes. Films were obtained by solvent evaporation. Cumulus cells obtained before hyaluronidase exposure were placed in culture onto PVA-BTCA hydrogel for cytotoxicity and cell attachment test. Eleven GV and 4 denuded oocytes and cumulus cells were added to PVA-BTCA culture system for IVM, at 38o.C and 5%CO2.

Results

Two replicates of cumulus cells exposure to PVA-BTCA culture system showed that the hydrogel allows for cell survival and adhesion to the substrate, after gentle swirling of the culture dish and change of the medium with the unattached cells, as observed under contrast phase microscopy. Having confirmed the hydrogels supported cell survival, 11 GV and 4 MI oocytes obtained from five different patients were added to the culture system together with cumulus cells isolated from oocytes of the same women. Eight GV and 2 MI oocytes reached MII (73% and 50%, respectively), after 24 hrs culture in the PVA-BTCA /cumulus cells culture system.

Conclusion

The PVA/BTCA gel represents a promising new approach to improve IVM culture conditions, particularly for Fertility Preservation. The system may enhance the number of mature oocytes collected after superovulation or during ovarian tissue fragmentation for cryopreservation, when small follicles and immature oocytes are released. PVA-BTCA culture system may also be used for individual follicle growth.

Keywords

IVM, chemically defined culture, PVA.