Pregnancy is a complicated physiological process. The multifaceted regulation of maternal–fetal interface is of great importance for maintaining normal pregnancy and avoiding fetal rejection and secondary abortion. Previous studies have focused on the clinical features or pathological biomarkers of fetal rejection and abortion. However, no significant breakthrough has been made. Therefore, it is important to understand the molecular mechanisms of recurrent pregnancy loss (RPL) to identify potential therapeutic strategies. The aim of this study was to investigate the pathogenesis of RPL. In this study, Relative and absolute quantitation (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis was used to identify differentially expressed proteins in decidual from RPL patients and matched normal controls. Further, Molecules NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3 (ndufb3) and cyclooxygenase-2 (COX-2) were validated by immunohistochemistry (IHC), Western blotting, CCK8 and mitochondrial red fluorescent probe (Mito-Tracker Red CMXRos). A total of 456 proteins reached the threshold of a 1.5-fold change were identified for further bioinformatics analysis. Upon mapping the differentially expressed proteins using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database, iTRAQ results were confirmed by assessing NDUFB3 and COX-2 protein levels in specimens of decidual tissue by Western blotting. Our study indicates that the level of COX-2 and NDUFB3 were significantly increased in decidual cell from RPL patients. Overexpression of NDUFB3 inhibited cell vitality and oxidative stress of decimal cell. Further, our found that overexpression NDUFBD3 in decidual cell decreased the mitochondrial membrane potential expression levels. These results suggest that NDUFB3 might play an important role in promote the pathological process of RPL. This comprehensive analysis of RPL proteomics reveals novel candidate: NDUFB3, which could be further investigated for explanation of the pathological mechanism of RPL.

中文翻译：

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复发性流产（RPL）患者蜕膜的蛋白质组学分析显示线粒体氧化应激功能异常

怀孕是一个复杂的生理过程。母胎界面的多方面调节对于维持正常妊娠并避免胎儿排斥和继发性流产至关重要。先前的研究集中于胎儿排斥和流产的临床特征或病理生物标志物。但是，没有取得重大突破。因此，重要的是要了解复发性流产（RPL）的分子机制，以确定潜在的治疗策略。这项研究的目的是调查RPL的发病机理。在这项研究中，将相对定量和绝对定量（iTRAQ）技术与液相色谱-串联质谱（LC-MS / MS）分析相结合，用于鉴定RPL患者和匹配的正常对照组蜕膜中的差异表达蛋白。此外，通过免疫组织化学（IHC），蛋白质印迹，CCK8和线粒体红色荧光探针（Mito-Tracker Red CMXRos）验证了分子NADH脱氢酶[泛醌] 1β亚复合物亚基3（ndufb3）和环加氧酶2（COX-2）。鉴定出总共456种蛋白质达到1.5倍变化阈值，以进行进一步的生物信息学分析。通过使用《京都议定书》的基因和基因组百科全书（KEGG）途径数据库对差异表达的蛋白质作图后，通过蛋白质印迹评估蜕膜组织标本中的NDUFB3和COX-2蛋白水平，证实了iTRAQ结果。我们的研究表明，RPL患者蜕膜细胞中COX-2和NDUFB3的水平显着增加。NDUFB3的过表达抑制了细胞活力和十进制细胞的氧化应激。进一步，我们发现在蜕膜细胞中过表达NDUFBD3降低了线粒体膜电位表达水平。这些结果表明NDUFB3可能在促进RPL的病理过程中起重要作用。对RPL蛋白质组学的全面分析揭示了新的候选物：NDUFB3，可以对其进行进一步研究以解释RPL的病理机制。