Developmental Potential And Cytogenetics Of Gonadotropin Stimulating And In Vitro Matured Mouse Oocytes

[BACKGROUND AND AIM] The gonadotropin stimulation (GS) ovulation protocols are widely applied in the field of assisted reproductive treatment of infertility, which can generate multiple follicles in order to collect more muture oocytes for in vitro fertilization (IVF). It is believed that more oocytes are collected and more embryos are available for embryo transfer (ET), which resulted in incresing the chance of pregnancy in each treatment cycle. However, more and more people worry about the GS side effects, such as increasing long-term risk of ovarian cancer and inducing OHSS etc. On the other hand, both patients and medical professionals pay high attention whether the quality of oocytes and subsequent embryonic developmental potential would be affected by high- dose use of gonadotropin stimulation.To decrease side effects of gonadotropin stimulation, researchers were interested in the techniques of in vitro maturation (IVM) of immature oocytes. Although about 60% to 70% of immature oocytes could reach nuclear maturity after in vitro mature culture, however, their fertilization rate and subsequent blastocyst formation rate are still lower compared with those of in vivo mature oocytes, indicating some quality defects of IVM oocytes. At present, most studies have focused on how to improve culture conditions for in vitro immature oocytes, but it is seldom to report the key factors, such as meiotic spindle organization and chromosome alignment as well as aneuploidy during MII stage, which determin IVM oocyte fertilization and subsequent developmental potential.The aim of this study was to explore the effcts of GS on oocyte fertilization and subsequent developmental potential; the effects of IVM on oocyte quality and subsequent developmental potential; the effects of GS and IVM on protein synthesis of MII oocytes; the effcts of GS on meiotic spindle organization and chromosome alignment of oocytes from in vivo and in vitro muturation and the effcts of GS and IVM on oocyte aneuploidy.[MATERIALS AND METHODS] 1. The experimental mice were randomly divided into four groups: a) natural cycle and in vivo matured oocytes (post-ovulation); b) stimulated cycle and in vivo matured oocytes (PMSG+hCG; post-ovulation); c) natural cycle and in vitro matured oocytes (pre-ovulation); d) stimulated cycle and in vitro matured oocytes (PMSG only; pre-ovulation). 2. immature oocytes were cultured in vitro by IVM technique and the mature rates of immature oocytes between natural cycle and stimulated cycle were compared. 3. IVF was carryed out to observe oocyte fertilization and the fertilization rate, formation rates of 2-cell embryo and blastocyst among four groups were compared. 4. one-direction SDS-PAGE electrophoresis and silver staining were employed to detect and compare the protein synthesis of matured oocytes at MII stage among four groups. 5. inverted microscope equipped with LC Polscope optics and controller combined with a computerized image analysis system was applied to observe and compare the oocytes meiotic spindle position and the spindle plate as well as the length of spindle plate among four groups. 6. immunofluorescent assay was performed to observe the meiotic spindle organization and chromosome alignment among four groups under fluorescent microscope. 7. Giemsa staining and FISH were applied to analyze the oocyte karyotypes among the four groups, respectively.[RESULTS] 1. There were no significant differences in all the parameters includingfertilization rates and the rates of 2-cell embryo and blastocyst formation of in vivo matured oocytes between gonadotropin stimulated and naturally cycling mice (P>0.05). 2. The fertilization and blastocyst formation rates of in vitro matured oocyte were significantly lower than those of in vivo matured oocyte derived from gonadotropin stimulated mice (P<0.05). 3. The fertilization and blastocyst formation rates of in vitro matured oocyte were significantly lower than those of in vivo matured oocyte derived from natural cycle mice (P<0.05). 4. There were no significant differences in the rates of nuclear maturation and fertilization of in vitro matured oocytes between gonadotropin stimulated and naturally cycling mice. However, the blastocyst formation rate of in vitro matured oocytes from gonadotropin stimulated mice was significantly higher than those from naturally cycling mice. 5. There were no significant differences in protein synthesis in oocyte cytoplasm during maturation between GS and IVM groups using SDS-PAGE/Silver-staining. 6. There were no significant differences in the length of spindle plates, meiotic spindle organizations, chromosome alignment and aneuploidy among the four groups. [CONCLUSIONS] 1. GS has no effct on the fertilization and subsequent developmentalpotential of in vivo matured oocytes. 2. IVM oocytes decrease their fertilization and developmental competence. 3. GS did not improve post-fertilization development of in vivo matured oocytes, but was beneficial to post-fertilization development of in vitro matured oocytes. 4. GS and IVM may not affect protein synthesis in oocyte cytoplasm during maturation. 5. GS and IVM did not increase the abnormal incidence of spindle organization and chromosomal alignment as well as chromosome aneuploidy: 6. There seems to be no direct correlation between lower developmental potential of IVM oocytes and incidences of meiotic spindle organization, chromosomal alignment and chromosome aneuploidy.