【摘要】：體外受精(in vitro fertilization, IVF)是一項(xiàng)應(yīng)用最為廣泛的輔助生殖技術(shù)(assisted reproductive technology, ART)。卵母細(xì)胞質(zhì)量和精子濃度在精卵結(jié)合過程中起到至關(guān)重要的作用,然而由于一些疾病或是自身排卵原因,亦或是取卵過程中人為操作的原因,臨床上經(jīng)常會獲得無卵丘包裹的卵母細(xì)胞稱為裸卵,(Denuded Oocyte, DO)甚至是無透明帶的卵母細(xì)胞(Oocyte Zona pellucida Free, OZ-)。雖然圍繞卵丘-卵母細(xì)胞復(fù)合體(cumulus-oocyte complex, COC)開展了許多研究,但對于DO或OZ-的卵子能否受精,以及受精的最佳精子濃度等問題,尚無系統(tǒng)報道。本研究以小鼠為實(shí)驗(yàn)對象,獲得體內(nèi)成熟的COC,用透明質(zhì)酸酶去除卵丘獲得DO,以及利用酸性M2操作液去除透明帶獲得OZ-,對于這三種在結(jié)構(gòu)上逐步遞減的卵母細(xì)胞進(jìn)行了如下實(shí)驗(yàn),以確定受精時的最佳精子濃度：1：將同一類型的卵母細(xì)胞與不同濃度的精子混合,確定每一類型卵母細(xì)胞的最佳受精精子濃度；2、將同一濃度的精子與不同類型的卵母細(xì)胞混合,以確定每一精子濃度梯度最適合的卵母細(xì)胞類型。我們的實(shí)驗(yàn)結(jié)果顯示：對于體內(nèi)成熟的COC,IVF最適精子濃度為106個/ml；對于DO,IVF最適精子濃度為107個/ml；而對于體內(nèi)成熟的OZ-,IVF最適精子濃度為107個/m1。精子濃度為103個/ml或104個/ml時,體內(nèi)成熟的DO獲得最高的受精率和胚胎發(fā)育率；精子濃度為105個/ml或106個/ml時,體內(nèi)成熟的COC獲得最高的受精率和胚胎發(fā)育率；精子濃度為107個/ml時,OZ-獲得最高的受精率。我們使用以上最適濃度的精子分別使COC、DO、OZ-體外受精后,獲得的囊胚免疫熒光染色統(tǒng)計囊胚細(xì)胞數(shù)和直徑,測量的結(jié)果表明COC組與DO組間沒有顯著差異,這兩組顯著高于OZ-組。綜上所述,COC在106個/ml精子濃度下受精率和胚胎發(fā)育率最高,但是如果不能獲得足夠的COC,可以使用DO進(jìn)行體外受精,DO的最適精子濃度為107個/m1。因?yàn)镺Z-后期的胚胎發(fā)育率極低,所以不建議使用OZ-用于體外受精操作。
[Abstract]:In vitro fertilization (in vitro fertilization, IVF) is one of the most widely used assisted reproductive technology (assisted reproductive technology, ART). The quality of oocytes and the concentration of sperm play a vital role in the process of spermatozoa binding. However, due to some diseases or self-ovulatory reasons, or because of human manipulation in the process of ovulation, Oocytes without cumulus are often obtained clinically, called naked oocytes, (Denuded Oocyte, DO) or even pellucidal oocytes (Oocyte Zona pellucida Free, OZ-). Although many studies have been carried out around the cumulus oocyte complex (cumulus-oocyte complex, COC), there are no systematic reports on whether the eggs of DO or OZ- can be fertilized and the optimal sperm concentration of fertilization. In this study, mature COC, was obtained from mice to obtain DO, by removing cumulus with hyaluronidase, and OZ-, was obtained by removing pellucidal zone from acid M2 operation solution. The following experiments were carried out to determine the best sperm concentration during fertilization: 1. Mix the same type of oocytes with different concentrations of sperm. Determine the optimal fertilization sperm concentration for each type of oocyte; 2. The same concentration of spermatozoa was mixed with different types of oocytes to determine the most suitable type of oocytes for each sperm concentration gradient. Our results show that the optimal sperm concentration for mature COC,IVF is 106 / ml; for DO,IVF and 107 / ml; for mature OZ-,IVF. When the sperm concentration was 103 / ml or 104 / ml, the mature DO obtained the highest fertilization rate and embryo development rate, while the mature COC obtained the highest fertilization rate and embryo development rate when the sperm concentration was 105 / ml or 106 / ml. When sperm concentration was 107 / ml, OZ- obtained the highest fertilization rate. The number and diameter of blastocyst cells were counted by immunofluorescence staining of blastocysts after in vitro fertilization of COC,DO,OZ- with the optimal concentration of spermatozoa. The results showed that there was no significant difference between COC group and DO group. These two groups were significantly higher than OZ- group. In conclusion, the fertilization rate and embryo development rate of COC were the highest at 106 / ml sperm concentration, but if enough COC, could not be obtained, DO could be used for in vitro fertilization, and the optimal sperm concentration of DO was 107 / M1. OZ- is not recommended for in vitro fertilization because of the very low rate of late OZ- embryo development.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R714.8

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本文編號：2302654