Supplementary Material for: Cervical Alpha-Actinin-4 Is Upregulated in Women with Threatened Preterm Labor and Microbial Invasion of the Amniotic Cavity

<b><i>Objective:</i></b> To characterize the proteome profile of women with threatened preterm labor (PTL) below 34;0 weeks with and without microbial invasion of the amniotic cavity (MIAC) using mass spectrometry in the amniotic fluid (AF) and Western blot analysis in the cervical mucus and the vaginal fluid. <b><i>Subjects and Methods:</i></b> In the discovery phase, a case-control study including 8 women with MIAC and 7 without matched for gestational age at sampling was performed. Proteomic profile characterization was done using the LTQ VELOS Orbitrap mass spectrometer in the AF. In the validation phase, a selection of the proteins differentially expressed by mass spectrometry in the genital samples of a prospective cohort of 109 women was validated by Western blot analysis. <b><i>Results:</i></b> In the discovery phase, the mass spectrometry analysis identified a total of 444 proteins. Sixteen were chosen for validation, being involved in defense (calgranulin A, B, C, C-reactive protein), cytoskeletal remodeling (alpha-actinin-4 [ACTN-4], plastin-2, α2-antiplasmin, vitronectin), metabolism (cystatin-β, glucose 6 phosphate isomerase, glutathione S-transferase, prostaglandin D2 synthase, corticosteroid-binding globulin), and vascular (α1-antichymotrypsin, hemopexin, endosialin) pathways. In the validation phase, cervical ACTN-4 was the only significantly upregulated protein in women with MIAC with an odds ratio of 6.8 (<i>p</i> = 0.002). <b><i>Conclusions:</i></b> Cervical ACTN-4 was significantly upregulated in the group of women with PTL with MIAC.

<b><i>研究目的：</i></b> 本研究旨在通过对羊水（amniotic fluid, AF）进行质谱分析，以及对宫颈黏液与阴道液进行蛋白质印迹法（Western blot）分析，明确孕34周以下先兆早产（threatened preterm labor, PTL）且伴/不伴羊膜腔微生物侵袭（microbial invasion of the amniotic cavity, MIAC）的女性的蛋白质组学特征。<b><i>研究对象与方法：</i></b> 发现阶段采用病例对照研究设计，纳入8例羊膜腔微生物侵袭阳性者与7例按采样孕周匹配的阴性受试者，通过LTQ VELOS Orbitrap质谱仪对羊水样本完成蛋白质组学特征鉴定。验证阶段则对109名女性组成的前瞻性队列的生殖道样本中经质谱筛选得到的差异表达蛋白，采用蛋白质印迹法进行验证。<b><i>研究结果：</i></b> 发现阶段的质谱分析共鉴定出444种蛋白质，其中16种被选为验证靶点，这些蛋白分别参与免疫防御（钙粒蛋白A、B、C，C反应蛋白）、细胞骨架重塑（α-辅肌动蛋白-4 [ACTN-4]、丝束蛋白-2、α2-抗纤溶酶、玻连蛋白）、代谢调控（半胱氨酸蛋白酶抑制剂β、葡萄糖-6-磷酸异构酶、谷胱甘肽S-转移酶、前列腺素D2合酶、皮质类固醇结合球蛋白）及血管通路（α1-抗胰凝乳蛋白酶、血液结合素、内皮唾液酸蛋白）。验证阶段结果显示，宫颈组织中的ACTN-4是唯一在羊膜腔微生物侵袭阳性受试者中显著上调的蛋白，其比值比为6.8（<i>p</i> = 0.002）。<b><i>研究结论：</i></b> 伴羊膜腔微生物侵袭的先兆早产女性宫颈组织中，ACTN-4呈显著上调表达。