Study On In Vitro Maturation Of Oocytes

Background: In-vitro maturation (IVM) of human oocytes would be an attractive alternative to gonadotrophin stimulation for in-vitro fertilization (IVF). It could avoid the complications of gonadotrophin stimulation and reduce the cost of drugs. However, the efficiency of existing IVM technique is suboptimal because embryos resulting from IVM oocytes exhibit frequent cleavage blocks and low implantation rate. It's commonly considered that oocytes in vitro matured have cytoplasmic maturation insufficiency. The improvement of culture condition of IVM is essential to achieve better outcome of IVM. During the development of follicles, granulosa cells proliferate and differentiate under FSH control. Granulosa cells mediate FSH control on oocyte maturation and secrete several factors to maintain an appropriate rnicroenvironment for oocytes to mature. Therefor, granulosa cell conditioned medium may be benefit for oocytes maturation, and it has not yet been fully studied. Cortical granule (CG) is an unique type organelle of mammalian oocyte. During the maturation of oocyte, CGs shows a specific change in distribution. The generally accepted indicator of final cytoplasmic maturation is the line underneath oocyte membrane constituted by CGs. Microtubule form spindles during Mil stage. It plays an important role in polar body extrusion, pronuclear formation and and early cleavage. As a result, it is reasonable to consider the line under-9-cell membrane and the spindles are both important indicators to evaluate cytoplasmic maturation.Objectives: 1. To study the microenvironment of human matured follicles. 2. To study the function of granulosa cells. 3. To study the effects of granulose cells conditioned medium, EOF and IGF-1 on in vitro matured Kunming mouse oocytes. 4. To utilize CGs and microtubules as the markers of mature cytoplasm to assess the efficiency of different cultured systems.Methods: 1. The levels of E2, P, and T in follicular fluid of 13 patients received IVF or 1C SI were evaluated by radioimmunoassay. EOF and IGF-1 were separately assessed by ELISA and immunoradiometric assay. 2. Human granulosa cells isolated from follicular fluid of 16 patients received IVF or ICSI were cultured in TCM199 medium. Granulosa cells were examined to detect FSHR by immunobiochemistry assay and RT-PCR. 3. The levels of E2, P, T, IGF-1, and EGF in granulosa cells conditioned medium were evaluated after cultured with 75 mlU/ml FSH or lOOpg/ml GnRH-a for 24 hours. 4. Oocytes in germinal vesicle (GV) stage of Kunming mice were randomly divided into 3 groups according to different IVM culture media. The first medium was a control group, which contained FSH 75mIU/ml and E2 1 nM. The second medium included EGF Ing/ml and IGF-1 lOng/ml in addition to the control. Likewise, the last medium contained 50% granulosa cells conditioned medium. Oocytes were cultured for 16 or 18 hours, and only the oocytes underwent first polar body extrusion were collected for future study. 5.-10-IVM oocytes were fertilized in vitro, and the process of fertilization, early cleavage, and blastocyst formation were recorded. 6. Oocytes were fixed for immunofluorescence. Examination of CGs and microtubules were performed by FITC labeled Lens culinaris agglutinin (LCA) and and- fi -tubulin under confocal scanning lasermicroscopy (CSLM) respectively. 7. Six GVBD stage oocytes from 2 patients received ICSI were matured in vitro and in vitro fertilization. Results: 1. The mean hormone levels in follicular fluid of matured follicles were: E2 336.65?70.39ng/ml, P 18.51+5.05 jj. g/ml, T 20.93?28.17ng/ml, IGF-1 303.76?62.78ng/ml, and EOF 2.9815.17pg/ml. 2. Over 75% granulosa cells isolated from follicular fluid expressed FSHR, 5 in 6 cases displayed FSHR mRNA bands after RT-PCR. 3. Concentrations of E2, T, and IGF-1 in supernantants after co-cultured with 75mIU/ml FSH were 8.42 ?4.12pg/ml/1000cell, 150.56 1115.99pg/ml/1000cell, and 4.02?.74ng/ml/1000cell, respectively. Thehormone values were significantly higher than the cont...