STUDY QUESTION Is the protein l-arginine methyltransferase 3 (PRMT3)/asymmetrical dimethylarginine (ADMA)/nitric oxide (NO) pathway involved in the development of recurrent miscarriage (RM), and what is the potential mechanism? SUMMARY ANSWER Elevated levels of PRMT3 and ADMA inhibit NO formation in the decidua, thereby impairing the functions of trophoblast cells at the maternal–foetal interface. WHAT IS KNOWN ALREADY Decreased NO bioavailability is associated with RM. ADMA, an endogenous inhibitor of nitric oxide synthase (NOS), is derived from the methylation of protein arginine residues by PRMTs and serves as a predictor of mortality in critical illness. STUDY DESIGN, SIZE, DURATION A total of 145 women with RM and 149 healthy women undergoing elective termination of an early normal pregnancy were enrolled. Ninety-six female CBA/J, 24 male DBA/2 and 24 male BALB/c mice were included. CBA/J × DBA/2 matings represent the abortion group, while CBA/J × BALB/c matings represent the normal control group. The CBA/J pregnant mice were then categorised into four groups: (i) normal + vehicle group (n = 28), (ii) abortion + vehicle group (n = 28), (iii) normal + SGC707 (a PRMT3 inhibitor) group (n = 20) and (iv) abortion + SGC707 group (n = 20). All injections were made intraperitoneally on Days 0.5, 3.5 and 6.5 of pregnancy. Decidual tissues were collected on Days 8.5, 9.5 and 10.5 of gestation. The embryo resorption rates were calculated on Day 9.5 and Day 10.5 of gestation. PARTICIPANTS/MATERIALS, SETTING, METHODS NO concentration, ADMA content, NOS activity, expression levels of NOS and PRMTs in decidual tissues were determined using conventional assay kits or western blotting. PRMT3 expression was further analysed in decidual stromal cells, macrophages and natural killer cells. A co-culture system between decidual macrophages (DMs) and HTR-8/SVneo trophoblasts was constructed to study the roles of the PRMT3/ADMA/NO signalling pathway. Trophoblast apoptosis was analysed via Annexin V-fluorescein isothiocyanate/propidium iodide staining. CBA/J × DBA/2 mouse models were used to investigate the effects of SGC707 on embryo resorption rates. MAIN RESULTS AND THE ROLE OF CHANCE Our results show that NO concentration and NOS activity were decreased, but ADMA content and PRMT3 expression were increased in the decidua of RM patients. Moreover, compared with the normal control subjects, PRMT3 expression was significantly up-regulated in the macrophages but not in the natural killer cells or stromal cells of the decidua from RM patients. The inhibition of PRMT3 results in a significant decrease in ADMA accumulation and an increase in NO concentration in macrophages. When co-cultured with DMs, which were treated with SGC707 and ADMA, trophoblast apoptosis was suppressed and induced, respectively. In vivo experiments revealed that the administration of SGC707 reduced the embryo resorption rate of CBA/J × DBA/2 mice. LIMITATIONS, REASONS FOR CAUTION All sets of experiments were not performed with the same samples. The main reason is that each tissue needs to be reserved for clinical diagnosis and only a small piece of each tissue can be cut and collected for this study. WIDER IMPLICATIONS OF THE FINDINGS Our results indicate that the PRMT3/ADMA/NO pathway is a potential marker and target for the clinical diagnosis and therapy of RM. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Key Research and Development Program of China (2017YFC1001401), National Natural Science Foundation of China (81730039, 82071653, 81671460, 81971384 and 82171657) and Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015). The authors have declared no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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巨噬细胞中不对称二甲基精氨酸和蛋白质 l-精氨酸甲基转移酶 3 介导的一氧化氮含量降低诱导滋养层细胞凋亡：复发性流产的潜在原因

研究问题 蛋白质 l-精氨酸甲基转移酶 3 (PRMT3)/不对称二甲基精氨酸 (ADMA)/一氧化氮 (NO) 通路是否参与复发性流产 (RM) 的发展，其潜在机制是什么？总结 答案 PRMT3 和 ADMA 水平升高会抑制蜕膜中 NO 的形成，从而损害母胎界面滋养层细胞的功能。已知情况 RM 的生物利用度降低。ADMA 是一种一氧化氮合酶 (NOS) 的内源性抑制剂，源自 PRMT 对蛋白质精氨酸残基的甲基化，可作为危重病死亡率的预测因子。研究设计、规模、持续时间共招募了 145 名患有 RM 的女性和 149 名接受选择性终止早期正常妊娠的健康女性。九十六名女CBA/J，包括24只雄性DBA/2和24只雄性BALB/c小鼠。CBA/J×DBA/2交配代表流产组，CBA/J×BALB/c交配代表正常对照组。然后将 CBA/J 怀孕小鼠分为四组：（i）正常 + 载体组（n = 28），（ii）流产 + 载体组（n = 28），（iii）正常 + SGC707（PRMT3 抑制剂）组 (n = 20) 和 (iv) 流产 + SGC707 组 (n = 20)。所有注射均在妊娠第 0.5、3.5 和 6.5 天进行腹膜内注射。在妊娠第 8.5、9.5 和 10.5 天收集蜕膜组织。在妊娠第 9.5 天和第 10.5 天计算胚胎吸收率。参与者/材料、设置、方法 NO 浓度、ADMA 含量、NOS 活性、蜕膜组织中 NOS 和 PRMT 的表达水平使用常规测定试剂盒或蛋白质印迹法测定。在蜕膜基质细胞、巨噬细胞和自然杀伤细胞中进一步分析 PRMT3 表达。构建蜕膜巨噬细胞 (DM) 和 HTR-8/SVneo 滋养细胞的共培养系统，研究 PRMT3/ADMA/NO 信号通路的作用。通过膜联蛋白V-异硫氰酸荧光素/碘化丙啶染色分析滋养层细胞凋亡。CBA/J × DBA/2 小鼠模型用于研究 SGC707 对胚胎再吸收率的影响。主要结果和机会的作用我们的结果表明，RM患者蜕膜中NO浓度和NOS活性降低，但ADMA含量和PRMT3表达增加。此外，与正常对照组相比，PRMT3 表达在巨噬细胞中显着上调，但在 RM 患者蜕膜的自然杀伤细胞或基质细胞中没有。PRMT3 的抑制导致 ADMA 积累显着减少和巨噬细胞中 NO 浓度增加。当与用 SGC707 和 ADMA 处理的 DM 共培养时，滋养层细胞凋亡分别受到抑制和诱导。体内实验表明，SGC707 的施用降低了 CBA/J × DBA/2 小鼠的胚胎再吸收率。限制、谨慎的原因 并非所有组的实验都使用相同的样品进行。主要原因是每个组织都需要保留用于临床诊断，并且每个组织只能切割和收集一小块用于本研究。研究结果的更广泛意义 我们的结果表明，PRMT3/ADMA/NO 通路是 RM 临床诊断和治疗的潜在标志物和靶标。研究经费/竞争兴趣(S) 本研究得到国家重点研发计划（2017YFC1001401）、国家自然科学基金（81730039、82071653、81671460、81971384和82171657）和上海市医学卫生学科的支持建设工程（2017ZZ02015）。作者声明没有利益冲突。试用注册号 不适用。国家自然科学基金（81730039、82071653、81671460、81971384、82171657）和上海市医学卫生学科建设项目（2017ZZ02015）。作者声明没有利益冲突。试用注册号 不适用。国家自然科学基金（81730039、82071653、81671460、81971384、82171657）和上海市医学卫生学科建设项目（2017ZZ02015）。作者声明没有利益冲突。试用注册号 不适用。