【作者】 徐岚；

【导师】 黄天华；

【作者基本信息】 汕头大学 ， 病理与病理生理学， 2008， 博士

【摘要】 [研究背景与目的]采用促性腺激素刺激(gonadotropin stimulation,GS)排卵是临床不孕症辅助治疗过程中常规使用的方案,目的是为了获得更多的卵母细胞用于体外受精,从而获得较多的胚胎供于移植,以期提高临床妊娠率。然而促排卵治疗过程中出现的药物副作用,如增加远期卵巢癌发生风险,诱发卵巢过度刺激综合征(ovarian hyperstimulation syndrome,OHSS)等越来越引起人们对母体使用促排卵药物安全性的担忧。此外,使用大剂量促性腺激素是否影响卵母细胞受精及胚胎发育潜能也引起人们极大关注。为了减少促排卵治疗带来的各种副作用,近些年来,采用卵母细胞体外成熟培养(invitro maturation,ⅣM)技术替代促排卵治疗已成为辅助生殖领域的研究热点。虽然目前未成熟卵母细胞经体外培养后细胞核的成熟率可以达到60%-70%,然而其受精率及形成囊胚的比例仍很低,说明体外成熟的卵母细胞可能存在质量缺陷。目前,很多研究集中在如何改善、优化体外培养条件,而对于决定ⅣM卵母细胞受精及其后胚胎发育的关键因素,如决定卵母细胞能否正确分裂的纺垂体微管结构以及与胚胎发育密切相关的染色体非整倍性等研究尚不多见。本课题以小鼠为模型,目的在于研究:①GS对卵母细胞受精能力及其后发育潜能的影响;②ⅣM对卵母细胞质量及其后发育潜能的影响;③GS和ⅣM对MⅡ期卵母细胞蛋白合成的影响;④GS对体内成熟(PMSG+hCG)和体外培养成熟(PMSG/不加hCG)卵母细胞的纺垂体结构和染色体排列的影响;5.GS和ⅣM对卵母细胞非整倍性的影响。[材料与方法]1.实验小鼠随机分为4组:自然周期卵母细胞体内成熟组;自然周期卵母细胞体外成熟组;促性腺激素刺激周期卵母细胞体内成熟组(PMSG+hCG);促性腺激素刺激周期卵母细胞体外成熟组(PMSG/不加hCG);2.采用ⅣM培养未成熟卵母细胞,并比较来自自然周期和促性腺激素刺激周期(PMSG/不加hCG)未成熟卵母细胞体外成熟率;3.通过体外受精技术观察各组成熟卵母细胞的受精情况,并比较各组受精率、2-cell胚和囊胚形成率;4.采用单向SDS—PAGE电泳/银染技术测定并比较各组成熟卵母细胞MⅡ期胞浆蛋白合成;5.采用LC PolScope进行活体观察各组成熟卵母细胞纺垂体和第一极体的位置关系,并测量和比较纺垂体出现率及长度;6.采用免疫荧光染色技术和荧光显微镜观察纺垂体的结构和染色体的排列;7.采用Giemsa染色技术结合FISH检测并比较各组成熟卵母细胞染色体数目异常。[结果]1.GS和ⅣM对小鼠卵母细胞受精及其后发育潜能的影响(1)促性腺激素刺激周期和自然周期体内成熟卵母细胞的受精率、2-cell胚和囊胚形成率相比无明显差异;(2)促性腺激素刺激周期体外成熟卵母细胞与体内成熟卵母细胞相比,受精率和囊胚形成率明显降低;(3)自然周期体外成熟卵母细胞与体内成熟卵母细胞相比,受精率和囊胚形成率明显降低;(4)促性腺激素刺激周期与自然周期体外成熟卵母细胞的核成熟率和受精率相比无明显差异,但前者的囊胚形成率明显高于后者。2.GS和ⅣM对MⅡ期卵母细胞蛋白合成的影响通过SDS-PAGE单向电泳/银染和凝胶成像系统分析,促性腺激素刺激和体外成熟培养对卵母细胞MⅡ期蛋白质合成无明显影响。3.GS和ⅣM对卵母细胞的纺垂体结构和染色体排列的影响通过LC Polscope活体观察及FITC/DAPI免疫染色结合荧光显微镜观察,结果表明促性腺激素刺激和体外成熟培养对卵母细胞MⅡ期纺垂体出现率、纺垂体长度、纺垂体结构形态和染色体排列无明显影响。4.GS和ⅣM对卵母细胞染色体非整倍性的影响通过Giemsa染色结合FISH技术检测各组成熟卵母细胞染色体数目,四组相比染色体非整倍性发生率无明显差异。[结论]1.GS不影响体内成熟卵母细胞受精及其后的发育潜能;2.ⅣM卵母细胞受精和其后的发育潜能降低;3.GS可以增加体外成熟卵母细胞受精后的胚胎发育潜能;4.GS和ⅣM不影响小鼠卵母细胞MⅡ期的蛋白合成;5.GS和ⅣM不增加卵母细胞纺垂体结构和染色体排列异常的发生率;6.GS和ⅣM不增加卵母细胞染色体非整倍性的发生;7.ⅣM卵母细胞的低发育潜能与纺垂体结构和染色体排列异常及染色体非整倍性的发生没有直接关系。更多还原

【Abstract】 [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.更多还原