Expression Of TFAM In Granulosa Cells Assess IVF Oocyte Maturation And Embryonic Development

Objective:In recent years,the increasingly high incidence of infertility has become a medical issue that not only hazards human life and health but also impedes the continuous development of human society.In vitro fertilization-embryo transfer(IVF-ET),a currently used treatment for infertility,oocyte fertilization,embryo and fetal development are affected by oocyte maturation and quality.Therefore,how to improve the oocyte maturity to increase the embryo quality is a hot research subject.Mitochondria are the most important organelles in the cell,and play an important role in the maturation of oocytes.Mitochondrial transcription factor A(TFAM)is the most significant regulator of mitochondrial DNA transcription and replication.Its identification of and binding to a specific sequence on mitochondrial DNA(mtDNA)promote mtDNA gene transcription and replication,thus preserving the integrity of mitochondrial oxidative respiratory chain function,providing energy for further oocyte development and contributing to fertilization and embryo development.The main purpose of this study was to investigate the expression of TFAM in IVF-ET patients,and to determine its effect on 2 pronuclei(2PN)rate,fertilization rate,egg utilization rate and pregnancy rate.We hope a further study on the impact of TFAM expression on oocyte maturation and continuous embryo development may increase clinical pregnancy rate,providing a new avenue for improving IVF pregnancy outcome.Methods:The study object: 85 patients who received IVF treatment for infertility at the reproductive department in No.4 Hospital of Hebei Medical University were included in this study.The inclusion criteria were: 1.using long-acting GnRH-a protocol,2.aged between 25-37,3.normal menstruation cycle,4.abaseline number of sinus follicles ? 7,5.a baseline level of FSH at 5-10 ng /ml.The specimen collection: granular cells were collected from the clear follicular fluid obtained in the first mature egg collected by needle aspiration guided by ultrasound on egg collection date.The follicular fluid were then placed on centrifuge tube(2000r)for 10 minutes.The collected granular cells were used for three purposes: 1.for detecting the expression of TFAM gene in granular cells by immunocytochemistry,2.for determining the expression of TFAM mRNA using reverse transcription-PCR(RT-PCR)3.for measuring the expression of TFAM protein using western blot.Statistical analysis:measurement data were collected using mean value± standard deviation(means ± SD).Data comparison was done using Student's t test.P<0.05 was considered statistically significant.SPSS 21.0(SPSS,Chicago,IL,USA)was used for statistical analysis.Result:1 Immunocytochemistry showed that TFAM was expressed in granulosa cells and was localized in cytoplasm.TFAM had no expression in nucleus.(Figure 1)2 There is a linear correlation between TFAM mRAN expression and protein expression in granulosa cells.(Fig.2?Fig.3?Fig.4)3 TFAM mRNA expression and IVF laboratory data.According to the ternary method,patients in the study were divided into three groups using the 33% and 76% of the TFAM mRNA relative expression:30 patients whose TFAM mRNA expression was lower than 33% of the relative expression were assigned to low expression group(TFAM m<0.3),27 patients between 33% to 76% were assigned to moderate expression group(TFAM 0.3-0.6)and 28 patients higher than 76% were assigned to high expression group(TFAM >0.6).There is no significant difference between the two groups of age,BMI,basal FSH,basal LH,GN and GN days.(Table 1)The egg utilization rate,2PN rate,fertilization rate and pregnancy ratewas significantly higher in the moderate expression group than in the other two groups(P <0.05).(Table 2)Conclusion:1 TFAM was expressed in the cytoplasm of human ovarian granular cells.2 The relationship between TFAM mRNA expression and protein expression is linear.3 There is a threshold of TFAM expression.A expression level higher or lower than that threshold will affect the mitochondrial transcription function,reducing the energy supply of mitochondria,and thereby affecting the fertilization rate and pregnancy outcome.Moderate expression of TFAM patients can achieve a better outcome of IVF.