Protocol standardization of granulosa cells culture, with esteroidogenic pattern characteristic of follicular phase of the menstrual cycle, for co-culture and oocytes in vitro maturation in Assisted Reproduction procedures

• Grant number: 09/04129-2
• Support Opportunities: Scholarships in Brazil - Master
• Start date: August 01, 2009
• End date: July 31, 2011
• Field of knowledge: Health Sciences - Medicine - Maternal and Child Health
• Principal Investigator: Ana Carolina Japur de Sá Rosa e Silva
• Grantee: Carolina Oliveira Campos
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

INTRODUCTION: Advances in the establishment of new culture systems for growth and differentiation of ovarian follicles and/or its components permits the investigation on basic aspects of follicular development and specific clinical situations, aiming the improvement of in vitro maturation techniques in Assisted Reproduction. There is already a well established granulose cell culture pattern that maintains the luteinizing process during culture, what turn it impossible to use this kind of culture as a co-culture medium for in vitro maturation of human immature oocytes. OBJECTIVE: The aim of this project is to develop a new model of culture medium that leads to luteinization complete or partial reversion and allows the use of these cells in co-culture of immature oocytes in comparison to the conventional granulose culture procedure. This reversion will evaluated by hormonal pattern production and ultra-structural characteristics.SUBJECTS AND MEYHODS: Human granulosa cells will be obtained during oocytes retrieval procedure in 10 patients submitted to Assisted Reproduction Technique (ART) in a Human Reproduction service. The cells will be extracted from follicular fluid and smeared in alfa MEM or TCM199 medium in a concentration of 30000cels/weel, and cultured for 144 hours. Culture medium will be changed every 48 hours and the removed medium stored in -20ºC for later analysis, thus medium will be collected with 48, 96 and 144 hours for estradiol and progesterone measurement. The final culture will be analyzed in optic microscopy and then resuspended and fixed for electronic microscopy. This project submitted and approved by the ethics committee.