To Explore The Mechanism Of Cell-free DNA Affecting The Embryo Quality

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.