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目的:探讨单卵母细胞所对应的卵丘颗粒细胞(cumulus cells,CCs)中生长分化因子-9(GDF-9)、骨形成蛋白-15(BMP-15)、转化生长因子β1(TGFβ1)、Smad3的表达与对应单卵母细胞质量及其发育潜能的关系以及在不同促排卵方案中的表达。方法:因男方因素或曾有过体外受精(IVF)不受精史接受卵胞质内单精子显微注射(ICSI)治疗的患者41例,单卵母细胞的CCs 332份(Gn RH-a组21例172份CCs,非垂体降调节组20例160份CCs)。拣卵后机械法剥离单卵子周围的CCs,将卵母细胞与收集的CCs一一对应进行分析。M_Ⅱ卵均采用ICSI授精,并观察其受精、卵裂及囊胚发育情况。采用Real-time PCR方法检测,比较各组CCs中GDF-9、BMP-15、TGFβ1和Smad3的相对含量。结果:1 CCs收集及PCR扩增产物可满足实验要求。2 Gn RH-a组及非垂体降调节组中,上述4种卵母细胞分泌因子,在正常受精组的相对含量均显著高于异常受精组;优质卵裂胚形成组的相对含量均显著高于非优质卵裂胚形成组;优质囊胚形成组中的相对含量显著高于非优质囊胚形成组。3 Gn RH-a组的CCs中BMP-15的表达量相对更高;而GDF-9、TGFβ1和Smad3在非降调节组的CCs中相对表达量较高。结论:1采用单卵母细胞的CCs的RNA进行研究,实际操作是可行的,RNA的质量和数量可达到实验要求。2 GDF-9、BMP-15、TGFβ1、Smad3可作为评价卵母细胞质量、预测卵母细胞发育潜能的客观指标。3降调节与否可能影响CCs中的卵母细胞分泌因子的表达水平,且与M_Ⅱ期卵母细胞发育潜能有关。
Objective: To investigate the expression of GDF-9, BMP-15, TGFβ1 in cumulus cells (CCs) , The relationship between the expression of Smad3 and the corresponding single oocyte quality and its developmental potential as well as the expression in different ovulation induction programs. METHODS: Totally 41 patients undergoing microscopic injections of intracytoplasmic sperm (ICSI) due to male factors or those who had had IVF insemination history were divided into three groups: 332 CCs of single oocytes (Gn RH-a group 21 172 cases of CCs, 20 cases of non-pituitary down-regulation group of 160 CCs). After picking eggs, the CCs around the single egg were dissected mechanically, and the oocytes were analyzed in one-to-one correspondence with the collected CCs. M_Ⅱ eggs were used ICSI insemination, and observed fertilization, cleavage and blastocyst development. Real-time PCR was used to detect the relative contents of GDF-9, BMP-15, TGFβ1 and Smad3 in each group of CCs. Results: 1 CCs collection and PCR amplification products to meet the experimental requirements. 2 Gn RH-a group and non-pituitary down-regulation group, the above four kinds of oocyte secretion factor, the relative content of the normal fertilization group were significantly higher than the abnormal fertilization group; high-quality cleavage embryo formation group relative content were significantly higher In non-high quality blastomere formation group, the relative content of high quality blastula formation group was significantly higher than that of non-high quality blastocyst formation group. The expression of BMP-15 in CCs of 3 Gn RH-a group was relatively higher, while that of GDF-9, TGFβ1 and Smad3 was higher in CCs of non-down-regulation group. CONCLUSION: 1 It is feasible to study the RNA of single-oocyte CCs. The quality and quantity of RNA can meet the experimental requirements. 2 GDF-9, BMP-15, TGFβ1, Smad3 can be used as objective indicators to evaluate oocyte quality and predict oocyte developmental potential. 3 down regulation or may affect the expression of oocyte secretion factors in CCs, and M_Ⅱ oocyte developmental potential.