The Role Of Microtubules And Microfilaments During In Vitro Maturation Of Human Oocytes And The Clinical Application Of Human Cumulus Cell Monolayer Co-Culture During In Vitro Maturation

SECTION 1 THE ROLE OF MICROTUBULES AND MICROFILAMENTS DURING IN VITRO MATURATION OF HUMAN OOCYTESOBJECTIVES:There are a series of biochemical changes in the cytoplasm during in vitro maturation (IVM) of human oocytes to insure the normal meiosis. It has been revealed that the organization of the cytoskeleton, in particular microtubules and micro filaments, plays an important role in the regulation of these dynamic events. The aim of the present study was to clarify the role of microtubules and microfilaments in several dynamic events, such as the condensation and movement of chromosomes, formation and migration of spindle, redistribution of cortical granule (CG) and mitochondria during IVM of human oocytes.METHODS:Germinal vesicle (GV) oocytes were collected from patients undergoing intracytoplasmic sperm injection (ICSI) treatment for male factors or combined with oviduct infertility from December 2007 to November 2008 at the Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University. During ovarian stimulation, GV stage oocytes were frequently obtained which were collected and donated to the present study after informed consent. GV stage oocytes were divided randomly into control and study groups. After cultured in drug-free maturation media (control group) or media containing lOμmol/L colchicine or cytochalasin B (CB, study group) for 48 h, chromatin state and position, spindle formation and migration, CG and mitochondria distribution were evaluated using confocal laser scanning microscopy.RESULTS:In the control group,68.42%(52/76) of the oocytes could develop to metaphaseⅡ(MⅡ), spindle locating at the cortical region and chromosomes aligning on the equatorial plane of the spindle. Before IVM, CG distributed evenly in the cytoplasm of human oocytes. Following IVM, it migrated to the cortical region, locating just beneath the oolemma in 73.53%(25/34) of the oocytes.In colchicine group, germinal vesicle breakdown (GVBD) could occur, chromosomes condensed and translocated to the cortex. However, spindles did not form; CG migrated to the cortex in 76.67%(23/30) of the oocytes arrested in metaphase I (MI) stage. Mitochondria maintained the peripheral distribution pattern in 47.06%(24/51) of the oocytes cultured for 48 h; only 37.25%(19/51) of the oocytes showed an even distribution pattern of mitochondria.In CB group, GVBD could occur but the meiotic process was arrested to MI stage in 83.33%(80/96) of the oocytes. Spindle formed and chromosomes condensed but their migration to the cortex was prohibited. CG could not migrate to the cortical region in 83.87%(26/31) of the oocytes. However, mitochondria could reach an even distribution pattern in 71.70%(38/53) of the oocytes.CONCLUSIONS:(1) GVBD and chromosomes condensation were not dependent on either microtubules or microfilaments. However, the migration of spindle and chromosomes to the cortical region was mediated by microfilaments during in vitro maturation of human oocytes; (2) Migration of CG to the cortex was mediated by microfilaments during in vitro maturation of human oocytes;(3) Redistribution of mitochondria to the inner cytoplasm was mediated by microtubules during in vitro maturation of human oocytes. SECTION 2 THE CLINICAL APPLICATION OF HUMAN CUMULUS CELL MONOLAYER CO-CULTURE DURING IN VITRO MATURATIONOBJECTIVES:Many attentions have been paid in recent years to IVM because of its special advantages. It now becomes one of the focuses in reproductive medicine. Compared to traditional in vitro fertilization (IVF) or ICSI, the rates of maturation, cleavage, and good quality embryo in IVM are still lower; the polyspermy rate is higher; the clinical pregnancy and implantation rates are lower. One of the important reasons is that the cytoplasm maturation is incompleted or not synchronous with nuclear maturation. Previous studies demonstrated that coculture with granulosa cell monolayer could improve maturation rate and developmental potential of oocytes. Our previous study also showed that coculture could improve cytoplasmic maturation rate of human oocyte. Based on previous studies, we further perform in vitro fertilization on human oocytes matured with granulosa cell monolayer to evaluate the effect of granulosa cell monolayer coculture on maturation and further development of human oocytes.METHODS:25 polycystic ovarian syndrome (PCOS) patients undergoing IVM/ICSI treatment for male factors or combined with oviduct infertility from November 2007 to October 2008 at Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University were included in the present study. Immature oocytes were cultured with granulosa cell monolayer for 28-30h. As control, oocytes from 70 patients in 94 cycles were cultured in traditional IVM medium. Then the rates of maturation, fertilization, cleavage, clinical pregnancy and implantation were evaluated.RESULTS:There were no significant differences in maturation and fertilization rates between the coculture and control groups (P>0.05). However, the cleavage rate was much higher in coculture group (P<0.05). Of the 20 transfer cycles in coculture group, 55 embryos were transferred and 7 (35.00%,7/20) clinical pregnancies were obtained, including 6 singleton and 1 twins. The implantation rate was 14.55%(8/55). Of the 75 transfer cycles in the control group,18 (24.00%,18/75) clinical pregnancies were obtained with 16 singletons and 2 twins. The implantation rate was 6.04%(20/331), which was much lower compared with the coculture group (P<0.05).CONCLUSIONS:Human cumulus cells monolayer co-culture could improve cleavage rate and the development potential of immature oocytes, which could further enhance implantation rate. It is beneficial and feasible to apply cumulus cells monolayer coculture during IVM in clinic routinely.