Theranostics 2021; 11(12):5813-5830. doi:10.7150/thno.58731

Research Paper

Extracellular vesicles derived from M1 macrophages deliver miR-146a-5p and miR-146b-5p to suppress trophoblast migration and invasion by targeting TRAF6 in recurrent spontaneous abortion

Jinli Ding1*, Yan Zhang2*, Xiaopeng Cai3*, Yi Zhang1, Sisi Yan1, Jiayu Wang1, Sainan Zhang1, Tailang Yin1✉, Chaogang Yang3✉, Jing Yang1✉

1. Reproductive Medical Center, Renmin Hospital of Wuhan University & Hubei Clinic Research Center for Assisted Reproductive Technology and Embryonic Development, Wuhan 430060, China.
2. Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan 430060, China.
3. Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
*These authors contributed equally to this work.

This is an open access article distributed under the terms of the Creative Commons Attribution License ( See for full terms and conditions.
Ding J, Zhang Y, Cai X, Zhang Y, Yan S, Wang J, Zhang S, Yin T, Yang C, Yang J. Extracellular vesicles derived from M1 macrophages deliver miR-146a-5p and miR-146b-5p to suppress trophoblast migration and invasion by targeting TRAF6 in recurrent spontaneous abortion. Theranostics 2021; 11(12):5813-5830. doi:10.7150/thno.58731. Available from

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Rationale: Emerging evidence demonstrates that insufficient migration and invasion of trophoblasts play critical roles in the pathogenesis of recurrent spontaneous abortion (RSA). Cell-to-cell communication at the maternal-fetal interface is essential to maintain the invasion and migration of trophoblasts. M1 macrophages, important immune cellular components at the maternal-fetal interface, have been reported to be elevated in decidua tissues from patients with RSA. Recent studies indicate that M1 macrophages modulate trophoblast biological behaviors; however, the underlying mechanisms remain poorly understood.

Methods: Extracellular vesicles (EVs) were isolated from the supernatant of M1 macrophages inducted from THP-1 cells (M1-EVs) by ultracentrifugation, identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting, and their miRNA profile was characterized by miRNA sequencing. Scratch wound healing and transwell assays were used to investigate the effect of M1-EVs on trophoblast migration and invasion. RT-PCR, western blotting, and luciferase reporter assays were conducted to uncover the underlying mechanism. Finally, animal experiments were employed to explore the effect of M1-EVs on embryo absorption in mice.

Results: M1 macrophages suppressed trophoblast EMT to reduce their migration and invasion abilities in vitro by secreting EVs. Through miRNA sequencing, miR-146a-5p and miR-146b-5p were identified as the most upregulated miRNAs in trophoblasts treated with M1-EVs. Further functional experiments showed that M1-EVs inhibited trophoblast migration and invasion by transferring miR-146a-5p and miR-146b-5p. Mechanistically, EV miR-146a-5p and miR-146b-5p inhibited EMT of trophoblasts by directly suppressing TNF receptor-associated factor 6 (TRAF6) expression at the post-transcriptional level. Furthermore, M1-EVs aggravated embryo absorption in mice. Clinically, expression of miR-146a-5p, miR-146b-5p, and TRAF6 were aberrant in placental villous tissues from patients with RSA, and negative correlations were found between miR-146a-5p/miR-146b-5p and TRAF6 expression levels.

Conclusions: Our findings indicate that miR-146a-5p and miR-146b-5p derived from EVs play important roles in intercellular communication between M1 macrophages and trophoblasts, illuminating a novel mechanism in M1 macrophage regulation of trophoblasts and their role in RSA.

Keywords: recurrent spontaneous abortion, M1 macrophages, trophoblasts, extracellular vesicles, TRAF6