METHODS: In total, 54 endometrial samples were collected during the proliferative and secretory phases from women with RPL (n = 28) and healthy controls (n = 26). Comprehensive proteomic and phosphoproteomic analyses were conducted using label-free liquid chromatography-tandem mass spectrometry (n = 44), and verified through Western blotting (n = 10). Three comparison groups were established: total RPL endometrium versus total control endometrium; RPL proliferative endometrium versus control proliferative endometrium; and RPL secretory endometrium versus control secretory endometrium.
RESULTS: Differentially expressed proteins and differentially phosphorylated proteins were identified in the three comparison groups. Combining pathway enrichment, network analysis and soft clustering analysis, the insulin/cyclic nucleotide signalling pathway and AMPK/mTOR signalling pathway were identified as the major contributors to the aberration of RPL endometrium. Western blotting verified altered expression of four proteins: cAMP-dependent protein kinase type I-β regulatory subunit, adenylate cyclase type 3, 5\'-AMP-activated protein kinase catalytic subunit α-2 and phosphatidate phosphatase LPIN2.
CONCLUSIONS: This exploratory study provides insights into the differentiated protein expression and phosphorylation profiles of the endometrium of women with RPL in both the proliferative and sectretory phases of the menstrual cycle. The results highlight potential proteins associated with the pathogenesis of RPL that may serve as potential indicators for RPL. The findings contribute to the identification of potential targets for RPL treatment as well as its pathogenesis.
方法:总的来说,在增生和分泌阶段,从RPL女性(n=28)和健康对照(n=26)收集了54个子宫内膜样品。使用无标记液相色谱-串联质谱(n=44)进行了全面的蛋白质组学和磷酸化蛋白质组学分析,并通过Western印迹验证(n=10)。建立了三个比较组:总RPL子宫内膜与总对照子宫内膜;RPL增生性子宫内膜与对照增生性子宫内膜;RPL分泌性子宫内膜与对照分泌性子宫内膜。
结果:在三个比较组中鉴定了差异表达的蛋白质和差异磷酸化的蛋白质。结合途径富集,网络分析和软聚类分析,胰岛素/环核苷酸信号通路和AMPK/mTOR信号通路被确定为RPL子宫内膜畸变的主要原因.Western印迹验证了四种蛋白质的表达改变:cAMP依赖性蛋白激酶I-β调节亚基,腺苷酸环化酶3,5'-AMP活化蛋白激酶催化亚基α-2和磷脂酸磷酸酶LPIN2。
结论:这项探索性研究为RPL女性在月经周期的增殖期和分离期子宫内膜的分化蛋白表达和磷酸化谱提供了见解。结果强调了与RPL发病机制相关的潜在蛋白质,这些蛋白质可以作为RPL的潜在指标。这些发现有助于确定RPL治疗的潜在靶标及其发病机理。