| Backgroud : Assisted reproductive technology (ART) has become an effective treatment for infertility for nearly three decades. To avoid multi-pregnancy, great efforts have been made to minimize number of embryos transferred. Since Single-embryo transfer resulted in a lower pregnancy rate.During the last 3 decades, morphological evaluation is the mostly used method to select embryos. Preimplantation genetic diagnosis (PGD) is the alternative, but is limited by its embryo-invassion. Hence, it is necessary to develop a non-invasive, objective and quantitative biomarker to choose the best embryo.Ovarian hyperstimulation is routinely used to increase the number of mature oocytes produced per cycle in human-assisted reproduction techniques. Disappointingly, implantation rates of in vitro fertilized (IVF) embryos are low, in contrast with high fertilization and cleavage rates. The majority of IVF embryos fail to implant, even fewer embryos develop to live birth. It has been reported that excessive ovarian stimulation is associated with a significant reduction in pregnancy rate and results in a shift in the window of receptivity in IVF cycles. Ovarian hyperstimulation caused low endometrial receptivity and possibly poor embryo quality. Little information is available about the genetic basis of the negative impact of gonadotrophin stimulation on embryo development. More extensive research is required to achieve a more whole understanding of the effection of super-ovulation on embryo, which provided biological research data for people to find effective biological marker for accurate evaluation of preimplantation embryos.Objectives: 1 To study the effects of superovulation on blastocysts, embryo implantation in vitro and in vivo by mice modle. 2 To study the effects of superovulation on genes express in mice blastocysts between the superovulated and control group. 3 To confirm the change of interested genes caused by superovulation at mRNA and proteins level.Methods: 1 The morphology of mice blastocysts in the superovulated group and the control groups were observed by microscope, then measure the diameters and evaluation grades of blastocyst. 2 We further used a blastocyst/uterine epithelial cell co-culture system to observe and compare blastocyst attachment, adhesion and outgrowth at 24h, 48h and 72h by microscopy in the superovulated group with the control groups. 3 Microarray hybridization was applicated to detect gene expression profiling in blastocyst of superovulated group and the control groups. The data were analyzed by hierarchical clustering analysis , gene ontogoly (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases.4 Gene expression profiles derived from microarray analysis were confirmed quantitatively by real-time RT-PCR analysis for which we randomly selected 14 genes. 5 At the end of the process, ELISA was performed to test the expressions of Il-6 protein in blastocyst culture medium. Results: First, we measured the diameter of blastocysts. The results showed that diameters of blastocysts from control group were significantly larger than those from the superovulation group (152.6±65.5 vs 145.3±76.9μm, p<0.001). Moreover, total cell number of blastocysts from control group was more than those from superovulation group (76.8±7.9 and 68.9±6.9μm, p<0.01). Mophological examination of the blastocysts revealed that there were more grade 3 blastocysts in the superovulation group than that in the control group (2.1±0.2 vs. 0.9±0.1 per mouse, p<0.01).We further used a blastocyst/uterine epithelial cell co-culture system to study the effects of superovulation on blastocyst implantation. Data were collected at different time (24, 48, and 72 h) after embryos transferred onto the monolayer of the uterine epithelial cells. Results showed that, compared to the control group, superovulation had little effect on blastocyst hatchment, attachment and outgrowth(p>0.05).Furthermore, A total of 180 genes were differentially expressed between the superovulated blastocysts and the natural ovulating blastocysts(≥1.5fold, p<0.01). Totally, 108 out of the 180 genes exhibited down-regulation and the remainder were up-regulated in the superovulation group. 5 secreted proteins encoded by differentially expressed genes ( IL-6, Efemp1, H2-K1, Ly96 and Tgfbi)were found, in which IL-6,Efemp1 and Tgfbi were associated with embryonic developmentThe fourth, 14 differentially expressed genes were randomly selected and conformed using qRT-PCR. The PCR results showed consistent change in gene expression as in the corresponding microarray experiments. Fold changes of all the tested genes were higher by qRT-PCR than results from the microarray analysis The last one, From 0.5dpc to 3.5dpc in vitro culture, we found that the content of IL-6 protein in blastocyst culture medium in the superovulation group was significantly less than that in the control group (3.973±1.392vs 7.642±1.317, P<0.01). The more the content of IL-6 protein were in blastocyst culture medium, the more blastocysts formed.Conclusions: These results indicated that superovulation have effect on embryo quality and development, which reflected in the reduced diameter of the blastocyst, blastocyst evaluation. But ovarian stimulation did not change the capacity of embryo implantation. Ovarian stimulation may mainly affect the inner cell mass and further affect the fetus developing to term. To our knowledge, It has been the first report that discovered a total of 180 genes were differentially expressed between the superovulated blastocysts and the natural ovulating blastocysts. Among them, 108 genes exhibited down-regulation and the remainder were up-regulated in the superovulation group. Then we randomly selected 14 genes for qRT-PCR, the result of qRT-PCR were consistent with that of the gene chip.We choose five genes (IL-6, H2-K1, ly96, Efemp1 and Tgfbi) that could encode secreted proteins from the differentially expressed genes, in which functions of IL-6, Efemp1 and Tgfbi were associated with embryonic development. We also selected IL-6, which mRNA was down-regulated by superovulation, to test the change at protein level in blastocyst culture medium. Using ELISA , we found that the content of IL-6 protein in blastocyst culture medium in the superovulation group was significantly less than that in the control group, which was consistent with the result that super-ovulation have reduced the level of IL-6 mRNA. At the same time we found that the higher level of IL-6 protein present in the blastocyst culture medium, the more blastocysts formed.In short, the discovery of differentially expressed genes caused by superovulation provides a possible mechanism underlying the negative effect of superovulation on embryo quality and development. The confirmed down-regulation of secreted protein IL-6 may help to develop a non-invasive evaluation method of preimplantation embryos and to improve the rate of pregnancy and reduce the rate of multiple pregnancy. |
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