Mechanisms Of In Vitro Maturation Of Oocytes

Objectives: 1.To investigate the effect of in vitro maturation and fertilization of oocytes and embryo transfer (IVM/F-ET) from unstimulated cycles in women with infertility due to polycystic ovary syndrome, and to find out what are the key points in the results of IVM/F-ET.2. To investigate the relationship between the developmental competence of the oocyte matured in vitro and apoptosis of granulosa cells surrounding the oocytes, in order to find out how to predict the potential developmental competence of oocytes matured in vitro by determining the rates of apoptosis in granulosa cells surrounding the oocytes, and the selection of high-grade embryo to transfer back to the uterus to increase the rate of implantation.3. To study the characteristic evaluation of oocytes under microscope and the relationship with the ability of development of immature oocytes; and the ultrastructure of human oocytes cultured in vitro was also obaserved.Material and Methods: 1. Forty-eight women with polycystic ovary syndrome (POCS) and insulin resistant (IR) were involved in this trial. Among them, 24 patients were selected out randomly to receive metaformen treatment for 3 monthes before IVM, the other 24 patients were given IVM treatment directly without any pretreatment. There were 92 cycles in total as the treatment group. Every cycle was given hCG 10000 IU 36h before oocyte retrieval. Immature oocytes were matured in vitro and fertilized by ICSI, and the resulting embryos were replaced back to the uterus. There were other 73 patients who had no symptoms of PCOS, received ICSI-ET treatment only because severe male factors as the control group. All the patients had their examination of basal endocrinology, OGTT and insulin level, IGF-I, IGFBP-1 and leptin level.) . 2. Examples were obtained from January 2006 to April 2006, in reproduction medical center of our hospital. Oocytes were retrieved by ultrasound-guided transvaginal immature follicle puncture. The immature human oocytes were collected and cultured in vitro for 32 hour, then the granulosa cells of every oocyte were collected by mechanical aspiration in HTF + 10%SSS containing 80IU/M1 hyaluronidase before intracytoplasmic sperm injection(ICSI). The rates of apoptosis in granulosa cells were analysed by terminal deoxynucleotidyl transferase mediated dUTp nick end labelling (TUNEL), and observed with light microscope, and compared with the scoring of immature oocytes before culture , the matured degree and subsequent fertilization and cleavage of the corresponding oocyte.3. Ultrastructure of oocytes at different stages and of different quality were observed by transmission electron microscope (TEM). Immature oocytes at germinal vesicle (GV) stage and at MI stage after germinal vesicle break-down before and after being cultured in vitro, as well as mature oocytes at MU stage after being cultured were evaluated according to their morphology of light microscope, and then studied by TEM.Results: 1. Compared with classical ICSI group, the maturation rate of oocytes (71.0% vs 86.9%), fertilization rate (66.1% vs 80.2%), cleavage rate (52.2% vs 74.4%), high quality embryo rate (24.4% vs 54.2%), and clinical pregnant rate (25.0% vs 34.7%) were all decreased in IVM treatment group significantly (P<0.05). Among all the examinations, only the level of leptin affected the results of IVM/F-ET significantly, that means low level of leptin meant high cilinical pregnant rate of IVM/F-ET. Pretreatment using metaformin can increase the pregnant rate of IVM/F-ET (P<0.05), but had no effect on fertilization rate and cleavage rate (P>0.05).2. The rates of apoptosis in granulosa cells of the two hundred and six oocytes were analysed. Before in vitro maturation, the oocytes were scored according to morphological characteristics, the rates of apoptosis in granulosa cells of different scoring oocytes were counted separately. We found that as the decreasing of the scoring of the oocyte, the rate of apoptosis in granulosa cells was increased. Among them, the average rate of apoptosis in granulosa cells of the 0'oocytes was much higher than the average rate of apoptosis in granulosa cells of the 3'oocytes and 4'oocytes (P<0.01). After in vitro maturation, 116 oocytes were matured (released the first polar body and reached MII stage ). As the decreasing of the matured degree of the oocyte, the rate of apoptosis in granulosa cells was increased, the rates of apoptosis in granulosa cells of oocytes degenerative or dead were the highest. Intracytoplasmic sperm injection(ICSI) was carried on 116 oocytes matured in vitro. The average rate of apoptosis in granulosa cells of oocytes which had ability to fertilize and form embryo was obvious decreased than the average rate of apoptosis in granulosa cells of oocytes which had not ability to fertilize and form embryo, As the decreasing of the developmental ability of the oocyte, the rate of apoptosis in granulosa cells was increased.3. High quality oocytes has more oportunity of fertilization and achieve high quality embryos (P<0.05). The number of granulosa cells (GC) is the symbol of the development of GC. The cleavge rate and high quality embryo rate in oocytes which had GC>6 were much higher than the oocytes which had GC<3 (P<0.05). At GV stage the microvilli on oocyte surface were rare and shorter. Nucleus with a dense nucleolus was distributed in the periphery of oocyte. After culture the oocyte microvilli became denser and increased in number. There was rich ooplasm with organelles, such as large numbers of secretion vesicles, mitochondria and lipid droplets in the GV oocyte. The end of cumulus cell expanded and penetrated into the oocyte zone, forming gap junctions with oocyte microvilli. Functional connection of the adjacent cumulus cells was by desmosomes. In mature oocytes the rough endoplasmic reticulum and Golgi apparatus decreased while the smooth endoplasmic reticulum increased in number. Mitochondria degenerated and lipid droplets fused to form bigger ones. Cortical granules were distributed beneath the cytoplasm membrane in a line manner. The cell junctions between oocytes and cumulus cells were interrupted. There were various organelles in the first polar body. The mature oocytes with lower quality appeared to have lots of swollen mitochondria with blurred cristae and membrane. Some of these oocytes had fractured first polar bodies and deformed zona pellucida. Their surrounding cumulus cells showed higher frequency of cell apoptosis.Conclusion: 1. IVM in unstimulated cycles is a feasible treatment for women with PCOS. Leptin level in the patients can affect the results of IVM/F-ET. As to the PCOS patients with IR, pretreatment with metaformin is a useful method to increase the clinical pregnant rate of IVM/F-ET.2. The relationship between the developmental ability of the oocyte and apoptosis of granulosa cells was closed. We can predict the developmental ability of the oocyte by determining the rate of apoptosis in granulosa cells.The functional state of granulosa cells had definite effect on whether the oocytes can mature after in vitro maturation and develop high-grade embryos or not.3. The oocytes which has high quality will develop better in vitro than the lower quality oocytes. The ultrastructure of human oocytes at different stages and of different quality cultured in vitro was revealed.