HCG日高孕酮水平患者子宫内膜容受性相关microRNA表达及其靶基因筛选与调控的相关研究

Implantation failure limited the development of assisted reproductive technology and the success of infertility treatment. The clinical data showed that higher progesterone concentration at HCG priming day in the serum of the superovulated women would affect the clinical pregnancy and IVF outcome, and moreover the ratio of these women was 35% of total women within the superovulated cycles. In spit of an important clinical issue, there are few of studies to discuss the potential molecular mechanism. In the present study, we focused on the 4 microRNAs identified by microRNA chip experiments between the endometrium samples from women with normal progesterone concentration and higher progesterone concentration at HCG priming day, and screened the target genes regulated by these 4 microRNAs. Furthermore these genes will be identified the functions in vitro and in vivo using the cell culture models and animal models, respectively. Finally these genes would be divided into two parts following the study results, one of them were differentiated expressed in these patients themselves, and the other part of them were differentiated expressed in the procedure of superovulation. In conclusion, the present study would discuss the molecular regulation mechanism between higher progesterone concentration at HCG priming day and the subsequent clinical pregnancy, and provided the academic proof for the solution of this clinical issue, and facilitate the study of the relation between hormones regulation and embryo implantation.

植入失败已经成为限制辅助生殖不孕症治疗成功的主要因素，而临床证据表明，接受促排卵治疗患者，HCG日孕酮水平过高会显著降低临床妊娠率，导致植入失败，更重要的是这部分患者占据整个接受促排卵治疗的患者人数的35%。尽管如此，对于这种HCG日高孕酮影响临床妊娠率的分子机制还不清楚。本研究拟利用前期研究筛选的HCG日高孕酮水平患者子宫内膜特异表达的4个microRNA，通过生物信息学方法筛选重要的靶基因，并在体外细胞水平与动物模型中进行进一步鉴定，研究这些基因在HCG日高孕酮水平患者中的作用，从而明确这部分基因是由于促排卵诱导发生表达差异的，还是在这部分患者本身就存在的。通过本研究，可以深入探索HCG日高孕酮与临床妊娠之间的分子调控机制，进而对该重要临床问题解决方案的提出提供理论依据，对于后续继续探讨激素调控改善辅助生殖成功率具有重要的价值和意义。

在辅助生殖技术开展过程中，取卵日血清孕酮升高患者的临床妊娠率和着床率降低，但是机制不清，导致缺少有效的解决办法，近年来研究报道，miRNA在调节子宫内膜容受性方面有重要作用，因此探索miR-125b等在体外受精-胚胎移植（IVF-ET）过程中HCG日高孕酮患者子宫内膜容受性中的作用，对于解释临床问题有重要意义。.本项目利用生物信息学软件分别预测miR451a、miR424、miR30b和miR125b的靶基因，并与mRNA芯片结果数据结合分析，找出相同或者出现频率较高的基因.进而收集子宫内膜标本，原代分离培养子宫内膜上皮和间质细胞并鉴定，检测miRNA在子宫内膜细胞中的表达水平。体外实验检测孕酮处理对子宫内膜细胞中miRNA表达变化的影响。接着细胞水平检测miR-125b对细胞迁移和侵袭的影响，动物水平检测miR-125b对胚胎着床的影响。重要结果及关键数据：1.在种植窗期，高孕酮患者子宫内膜上皮细胞中的miR-125b的表达显著高于非高孕酮患者，且体外培养的人子宫内膜上皮细胞中miR-125b的表达呈现孕激素依耐性，而人子宫内膜间质细胞中未发现类似变化。2.双荧光素酶报告基因试验证明MMP26是miR-125b的靶基因，体内体外试验表明MMP26与miR-125b的表达水平呈现相反的关系。3.在体外培养的人子宫内膜上皮细胞中过表达miR-125b显著抑制细胞的迁移和侵袭能力。4.在小鼠胚胎着床过程中，子宫内膜上皮细胞中miR-125b的表达在着床前期显著高于着床期和着床后期，且着床部位显著低于着床间部位。5.在小鼠子宫内膜上皮细胞中过表达miR-125b显著减少胚胎着床个数。该项目关于子宫内膜容受性与孕激素的机制研究，发现并验证了MiR-125b在其中的分子作用机制，为临床上制定高孕酮ART患者的胚胎移植策略提供了理论支持，为临床上推行冻融胚胎移植方案助力，打破了促排卵过程中若孕酮升高则建议放弃治疗的国际惯例，将此类患者的妊娠率提高30%左右。