人类胚胎分泌糖链及糖蛋白的筛选及其临床应用

In vitro fertilization - Embryo transfer (IVF-ET), it is the key point for the successful clinical pregnancy to select the embryos to transfer with the best quality and potential implantation. The embryo assessment mainly based on the morphological observation for now lacking the objective, quantitative, and noninvasive screening markers. In the previous study on the surrounding medium of human embryos by lectin microarray screening, it was found that human blastocyst may secrete specific glycan reflecting embryo development. Therefore, the study group will screen the specific glycan and glycoprotein in human embryonic secretome by technologies of glycomics and proteomics. The experimental group will consist of 100 cases of embryo culture medium from clinical pregnancy, while 100 cases of embryo culture medium without clinical pregnancy to be the contrast group. The specific glycan will be detected by lectin microarray detection, which is to assess the potential diagnostic value of embryo combining with clinical data analysis. Then the mass glycans and glycoproteome analysis system will be employed to identify the special glaycan and glycoprotein secreted from those successful implanted embyos. Analysis model will be built for the embryonic secreted glycoproteome. Finally, the ELISA kit will be built for selecting the embryos to transfer, which would be much helpful for the clinical application of quantizable embryonic biomarkers.

在体外受精-胚胎移植（IVF-ET）中挑选质量最好、最具有种植潜能的胚胎进行移植是成功临床妊娠的决定性因素。然而目前胚胎筛选主要依据形态学观察，缺乏客观定量的、无创的筛选指标。本研究组在前期研究中对培养液凝集素芯片筛查，发现人类囊胚可分泌特异性糖链，且与胚胎发育相关。因此拟从糖生物学角度利用糖组学和蛋白质组学技术，对胚胎分泌特异性糖链和糖蛋白进行筛选。拟收集成功临床妊娠和未临床妊娠的胚胎培养液各100例，应用凝集素芯片筛选培养液中胚胎分泌的特异性糖链和糖链谱变化，结合临床资料分析其预测胚胎种植潜能的诊断价值；应用基于质谱技术的糖组学和蛋白质组学分离鉴定胚胎分泌的特异糖链及糖蛋白，建立胚胎分泌糖蛋白组分析模型；建立胚胎分泌特异性糖链的凝集素ELISA试剂盒，为胚胎筛选提供客观量化检测指标以应用临床。

在体外受精-胚胎移植（IVF-ET）中挑选质量最好、最具有种植潜能的胚胎进行移植是成功临床妊娠的决定性因素。然而目前胚胎筛选主要依据形态学观察，缺乏客观定量的、无创的筛选指标。本研究通过IVF-ET后临床妊娠组和非临床妊娠组的囊胚培养液的样本采集和凝集素芯片筛查，共筛选出九个（AAL, NPA, UEA-I, MAL-I, LCA, PHA-E+L, GNA, DBA, BPL），通过磁珠-凝集素复合体提取凝集素（UEA-I、 LCA）对应的糖蛋白进行质谱鉴定。从蛋白质组学研究，根据发育结局分为囊胚形成组（A组）、发育停滞组（B组）和空白对照组（C组），每组收集104份培养液。通过非标记定量蛋白质组学技术（Lablefree）对三组囊胚培养液进行蛋白质组学分析，共计筛选出16个差异蛋白。在糖组结果和蛋白质组结果基础上，再结合相关文献，筛选出与胚胎发育、种植潜能相关的9种差异表达蛋白质进行进一步蛋白芯片的验证。通过分析各组的各蛋白质的平均含量的差异显著程度，最终发现6种含量显著差异且与囊胚发育质量显著相关的特异蛋白质，分别为APOA2、APOH、HBB、ORM1、RBP4和SERPING1（下图）。囊胚的质量与妊娠结局是紧密相关的，因此，推测囊胚培养液中的特异蛋白质组含量差异能反映该囊胚的种植结局，可以作为囊胚筛选的标志物。联合应用形态学评分和蛋白含量检测技术，可为囊胚筛选提供更加客观的标准。