【作者】 张文娟；

【导师】 王兴玲；

【作者基本信息】 郑州大学 ， 生殖医学， 2016， 硕士

【摘要】 随着不孕症患者的逐年增多,对于人类辅助生殖技术(ART)来说,如何提高临床妊娠率非常重要。而妊娠的达成需要较好的胚胎质量和优良的子宫环境。目前对于胚胎质量来说,较多的评价依赖于胚胎的形态学标准,然而,依靠胚胎形态学的主观观察来预测妊娠结局是受限制的。最近的许多研究都集中于研究来源于卵母细胞微环境的非侵入性生物学标志物,来提高胚胎选择的精确性。游离DNA(cf DNA),是游离核酸的一种,为双链DNA分子,且有着比基因组DNA更低的分子量,主要来源于坏死或者凋亡的进程。最早是1948年被Mendel和Metais在血浆中发现。近年来被广泛研究于肿瘤、妇产科等领域。多项研究发现血液中cf DNA水平在一些癌症和严重疾病的患者体内是升高的,基于此,cf DNA已经被用于非侵入性的生物学标志物来早期诊断和判断某些疾病的预后。而基于母体血液中胎儿cf DNA的非侵入的产前诊断检测,也构成了妇产科的一种很有前景的方法。目前,有研究发现卵泡液中的cf DNA水平与胚胎质量存在着负相关。如果卵泡液中的cf DNA含量确实与胚胎质量存在着密不可分的关系,应用其对胚胎质量进行非侵入性的预测将会是一种很有前景的方法。本研究通过对卵泡液中cf DNA含量与胚胎质量的关系探讨,首先验证了国外关于卵泡液中cf DNA水平与胚胎质量关系的研究,确实呈现负相关。进而通过cf DNA与氧化应激及凋亡的关系,进一步探讨cf DNA与胚胎质量存在关联性的原因。目的人卵泡液中cf DNA含量与胚胎质量之间是否存在相关性,并探讨cf DNA能够影响胚胎质量的原因。材料与方法1.实验材料:研究选取了从2014.09-2015.02于郑州大学第三附属医院生殖中心行新鲜周期体外受精(in vitro fertilization IVF)或卵胞浆内单精子显微注射技术(Intracytoplasmic sperm injection,ICSI)助孕治疗的189位患者,取卵日分别收集每位患者的所有卵泡液。入选患者纳入标准为:1.年龄≤40岁;2.体重指数:18-25kg/m2;3.月经周期21-35天,内分泌正常;4.未发现有卵巢或子宫器质性病变者;5.治疗方案为黄体中期短效长方案。剔除标准:女方不孕原因为多囊卵巢综合症、反复流产史、粘膜下层肌瘤,子宫粘连、子宫内膜异位症、HPV感染等的患者。2.方法:(1)分别使用酚氯仿异丙醇法(方法A)、改良酚氯仿异丙醇法(方法B)、Qiagen试剂盒法(方法C)量化其中48份卵泡液的cf DNA含量,得出最佳检测方法。余下的141份卵泡液选用最佳法进行检测;(2)在189份卵泡液中随机选取20份卵泡液,使用淋巴细胞分离液梯度分离出其中的颗粒细胞,将颗粒细胞随机分为四组,每组5例,分别加入不同浓度cf DNA进行共培养24h;(3)使用QRTPCR分别检测每组氧化应激相关因子的m RNA表达量;(4)使用Western-Blot检测加入cf DNA后不同时间段相关凋亡因子的蛋白表达量;(5)使用流式细胞仪分别检测每组的凋亡率。3.统计学方法:用SPSS17.0统计软件进行统计学分析,计数资料用t检验,计量资料用X2检验,相关分析用Spearman相关检验,P<0.05为差异有统计学差异。结果1.卵泡液中cf DNA提取方法比较的结果:酚氯仿异丙醇法(方法A)、改良酚氯仿异丙醇法(方法B)、Qiagen试剂盒法(方法C)这三种方法的OD值分别为(1.72±0.06),(1.69±0.05),(1.75±0.03),没有明显差异;方法A所测得的cf DNA含量(1.518±0.095mg/ml)明显低于方法B(1.825±0.114 mg/ml)和C(1.838±0.106mg/ml),差异有统计学意义(P<0.05),方法B和方法C效果相当,没有显著差异,但方法C花费较之方法B昂贵。2.优胚率与cf DNA的相关性分析:优胚率与cf DNA含量呈负相关,相关系数为-0.865,且P<0.05。3.氧化应激相关因子的m RAN表达量:FOXO1、TRAIL、Caspase-3、Fas L、Fas五个因子的m RAN表达量随着培养基中cf DNA浓度的升高(从0mg/ml到6mg/ml)逐渐升高,且与对照组比较,均有统计学意义(p<0.05)。4.死亡受体途径中相关凋亡因子的蛋白表达量:当培养基中cf DNA浓度为2mg/ml时,随着培养时间增加(0h、2h、4h、8h),活化的Caspase-8、Fas L、Fas的蛋白相对表达量增加,活化的Caspase-3虽然在培养时间2h时,蛋白相对表达量有稍许下降,但在随后4h、8h的检测时,其相对表达量呈上升趋势,且与对照组相比,均有统计学意义(p<0.05)。5.凋亡率的比较:当在培养基中加入cf DNA浓度分别为0mg/ml、2mg/ml、4mg/ml、6mg/ml时,共培养24h后,凋亡率分别为13%、24%、34%、48%,且与对照组相比,均有统计学意义(p<0.05)。结论1.改良酚氯仿异丙醇法(方法B)是三种方法中性价比最高的量化卵泡液中cf DNA的方法,而Qiagen试剂盒法是最为简单易操作的方法。2.cf DNA与优胚率负相关,可能是由于卵泡发生过程中cf DNA导致氧化应激,进一步触发颗粒细胞凋亡,使颗粒细胞凋亡率增高,卵母细胞质量下降,进而导致胚胎质量下降引起的。3.cf DNA可能是一个很有潜力的预测胚胎质量的非侵入性检测指标。更多还原

【Abstract】 With the increasing number of infertility patients, how to improve the clinical pregnancy rate is very important for human assisted reproductive technology. The pregnancy needs better embryo quality and superior uterine environment. Currently, the evaluation of embryo quality more dependents on the morphological criteria of the embryo. Nevertheless, the subjective observation of embryo morphology to predict a successful pregnancy shows limitations. Therefore many recent works have focused on the identification of new non-invasive biomarkers based on the analysis of the oocyte microenvironment to improve the accuracy of embryo selection. Circulating cell-free DNA(cf DNA) are double-stranded molecules with low molecular weight than genomic DNA, in the form of short fragments(between 70 and 200 base pairs in length) or long fragments up to 21 kb. Cf DNA result from apoptotic or necrotic processes. The presence of circulating cf DNA in human plasma was reported in 1948 by Mendel and Metais. In recent years, cf DNA has been widely studied in the fields of oncology, obstetrics and gynecology, etc. A number of studies found that the concentration of cf DNA was elevated in the patients’ blood with cancers or severe diseases. For that reason, cf DNA is used as non-invasive diagnostic and/or prognostic biomarker for some diseases. Similarly, the emergence of non-invasive prenatal testing, based on fetal cf DNA detection in the maternal blood,constitutes a promising approach in obstetrics and gynecology. At present, a study found there was a close negative correlation between cf DNA level and embryo quality. If cf DNA does have a close relationship with quality of the embryos, it will be a promising method to predict the embryo quality. In this study, the negative relationship between the content of cf DNA in follicular fluid and the quality of embryos was found. The study which was found by foreign countries of cf DNA and embryo quality was verified. Furthermore, through the study of the relationshiop between cf DNA and apoptosis and oxidative stress.we maybe can explore the reason of the relationship of cf DNA and embryo quality.Objective To verify the relationship between the content of cf DNA and good quality embryo rate. And to expolore the reasons that why the cf DNA can affect the embryo quality.Materials and Methods 1. Materials:the study selected 189 patients who were undergoing fresh cycle IVF or ICSI in the reproductive center of the Third Affiliated Hospital of Zhengzhou University from 2014.09-2015.02. Inclusion criteria:1.Age<40; 2. Body mass index:17-30kg/m2; 3. Menstrual cycle was 21-35 days with normal endocrine; 4. No ovarian or uterine implement qualitative sex pathological change. 5. Short gonadotropin-releasing hormone agonist(Gn RH-a) protocols. Remove: polycystic ovary syndrome(PCOS), recurrent abortion history, submucosal myoma, uterine adhesion, the application of small doses of aspirin, HPV infection, ect. 2. Methods: The follicular fluid of 189 patients were collected. 48 of follicular fluid was quantified using above phenol-chloroform- isoamyl alcohol extraction(Method A)、modified phenol – chloroform- isoamyl alcohol extraction(Method B)and Qiagen kit method(Method C)respectively. Then compare the cf DNA concentrations and OD260/280 ratio in order to obtain the most suitable method. The rest follicular fluid samples were detected by the optimal method. Then we selected 20 patients’ follicular fluid. Granular cells were isolated by gradient of lymphocyte separation. The granule cells were randomly divided into four groups, 5 cases in each group. We then added different content of cf DNA into the granule cells. In the last, the m RNA expressiong of some oxidative stress related factors were detected by QRTPCR in each group;the protein expression of apoptosis factors were detected by Western blot; the apoptosis rate was detected by flow cytometry. 3. Statistical methods: We used the one-way ANOVA for continuous parametric data and the X2 test for categorical variables. For paired continuous parametric data we used paired T test. Continuous parametric data were presented as mean±SD, and categorical variables as numbers and percentages. Results were considered significant when p<0.05.Results 1. Comparion of cf DNA extracgtion methods in follicular fluid: The OD260/280 of cf DNA by phenol-chloroform- isoamyl alcohol extraction(Method A)、modified phenol – chloroform- isoamyl alcohol extraction(Method B) and Qiagen kit method(Method C)were(1.72±0.06),(1.69±0.05),(1.75±0.03), there were no significance difference among the three methods. The content of cf DNA extracted by the three methods were(1.518±0.095mg/ml),(1.825±0.114mg/ml),(1.838 ± 0.106mg/ml).The concentrations of cf DNA measured by method A was significance lower than that of method B and C(P< 0.05). There were no significance difference among method B and C. The method B was the most suitable method because it cost less. 2. Correlation analysis of embryo rate and cf DNA: The good quality embryo rate and cf DNA content was negatively correlated. The correlation coefficient was-0.865. 3. The m RNA expression of oxidative stess related genes: the m RNA expression of Fox O1, TRAIL, Fas, Fas L, caspase-3 with the increasing of cf DNA concentration in medium culture(0mg/ml, 2mg/ml, 4mg/ml,6mg/ml) was gradually increased.What’s more, there was statistically significant between each group.(P<0.05). 4. The protein expression of apoptosis factors in Death receptor pathway: when the cf DNA content was 2mg/ml in cultured medium, the protein expression of cleaved caspase-8, Fas,Fas ligand(Fas L) was increased with the cultured time from 0h to 8h. Though the protein expression of cleaved caspase-3 was declined slightly in when cultured 2h,the protein expression was increased when cultured 4h and 8h compared with 2h. 5. Compared of apoptosis rate: When the cf DNA content was 0mg/ml, 2mg/ml, 4mg/ml, 6mg/ml,8mg/ml respectively in culture medium, the apoptosis rate was 13%, 24%, 34%,48% respectively.Conclusions 1. Method B was the highest cost-effective method. 2. The cf DNA can be a noninvasive marker for oocyte embryo qulity. 3. cf DNA concentration was negatively correlated with embryo quality. The reason maybe is that: cf DNA may cause oxidative stress in the process of follicle, which can further trigger apooptosis, increase the apoptosis rate, and in result decrease the quality of oocyte.更多还原