| Since the derivation of the first human embryonic stem cell (hESC) lines by Thomson and coworkers in1998, more than1,200different hESC lines have been established worldwide. Nevertheless, there is still a recognized interest in the establishment of new lines of hESC, particularly from HLA types and ethnic groups currently underrepresented among the available lines. The hESC are defined by unlimited self-renewal and the ability to differentiate—both in vitro and in vivo—into cell types of endodermal, mesodermal and ectodermal origins, rendering them a promising applicability in cell replacement therapies. These characteristics also make ESCs a powerful tool for studying the molecular mechanisms underlying cellular differentiation, as well as for accessing the biological effects of pharmaceutical compounds on the normal embryo development. However, we must face to the problem of source of the embryo in the process of the establishment of the hESC. Since Lerou’workgroup had successfully established the human embryonic stem cell lines with the poor morphology, low developmental potential embryos, there are a large number of researchers have focused on the use of discarded embryos for the establishment of human embryonic stem cell lines. That can avoid ethicallearn on the dispute. However, we still have to face the problem of low blastocyst formation rate of discarded embryos and poor quality blastocysts.Currently, there are some groups that have reported that group embryos culture to select the most appropriate embryonic development space and density can even effectively promote the further development of the embryo, and thus improve the clinical pregnancy rate. Similarly, we also found that the same phenomenon in animals (such as mice, sheep, cattle, etc.).The small molecules secreted by the embryos maybe promote the development of embryos and the potential to form the blastocyst. However, the composition of the substances is not very clear. Nowadays, there is not a system report of the group culture of the embryos. The purpose of this paper is that the grouping culture of the embryo in order to illustrate the effect on the developmental potential of discarded embryos. Thus, it is able to provide a reference for embryo culture in IVF/ICSI cycles. At the same time, it can allows us to use discarded embryos more efficiently to establish more human embryonic stem cell lines for research use.Objective:To explore the effect of group culture on the developmental potential of discarded embryos in IVF/ICSI cycles. To establish the human embryonic stem cell lines for the future research.Methods:1. Fresh discarded embryos were collected from the IVF/ICSI-ET program in the reproductive medical center of the first affiliated hospital of Zhengzhou university. All patients signed informed consent before experiment. Sequential culture was used for developing these embryos into blastocysts. According to the numbers of the embryos cultured together, the embryos were divided into groups.2. Mechanical method were used to isolate the inner cell mass (ICM) of blastocyst from the embryo. Then we inoculated the ICMs on feeder layer. Purification was done by repeated passage using the mechanical method. After identification of those cells, the hESCs lines were estblished.Results: 1. In this study, we collected1223fresh discarded embryos. These embryos were sequential cultured to the blastocysts. In total, we got221ICMs. The baseal informations of the patients are not different.2.1PN embryo’s blastocyst formation rate and quality embryo formation rate was significantly higher than the2PN and3PN embryos’(P<0.05). Compared with single embryo culture in1PN embryos, two and three embryos group cultivation can increase quality of blastocyst (P<0.05). In2PN embryos, compared to single embryo culture group,two and three embryos group cultivation can increase the blastocyst formation rate (P<0.05).At the same time, four embryos’blastocyst formation rate is less than three embryos group cultivation (P<0.05) with statistical significance. Compared with single embryo culture group, three embryo group cultivation increased quality blastocyst formation rate (P<0.05). At3PN groups, compared to single embryo, three embryos group cultivation increased formation rate (P <0.05).Also compared with three embryo culture group, two and four group cultivation were lower (P<0.05).3. In this study, we collected discarded embryos and developed them into blastocysts, used mechanical method to separate ICMs of high-quality blastocysts, planted the ICMs in the feeder layers which density was3.5x105/cm2. In total, we successfully established2hESCs lines. All of them had been through complete identification.Conclusion:1. The group culture of the embryos can improve the blastocyst formation rate and blastocyst quality to some extent. Three embryos group cultivate is the most appropriate number. Following, two group cultivation. Single and four group cultivation has no significant difference.2. The Fresh discarded embryos can be used to establish the hESCs lines. |
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