Intrauterine adhesions (IUA), characterized by endometrial fibrosis, is a common cause of uterine infertility. We previously demonstrated that partial epithelial-mesenchymal transition (EMT) and the loss of epithelial homeostasis play a vital role in the development of endometrial fibrosis. As a pro-survival strategy in maintaining cell and tissue homeostasis, macroautophagy/autophagy, conversely, may participate in this process. However, the role of autophagy in endometrial fibrosis remains unknown. Here, we demonstrated that autophagy is defective in endometria of IUA patients, which aggravates EMT and endometrial fibrosis, and defective autophagy is related to DIO2 (iodothyronine deiodinase 2) downregulation. In endometrial epithelial cells (EECs), pharmacological inhibition of autophagy by chloroquine (CQ) promoted EEC-EMT, whereas enhanced autophagy by rapamycin extenuated this process. Mechanistically, silencing DIO2 in EECs blocked autophagic flux and promoted EMT via the MAPK/ERK-MTOR pathway. Inversely, overexpression of DIO2 or triiodothyronine (T3) treatment could restore autophagy and partly reverse EEC-EMT. Furthermore, in an IUA-like mouse model, the autophagy in endometrium was defective accompanied by EEC-EMT, and CQ could inhibit autophagy and aggravate endometrial fibrosis, whereas rapamycin or T3 treatment could improve the autophagic levels and blunt endometrial fibrosis. Together, we demonstrated that defective autophagy played an important role in EEC-EMT in IUA via the DIO2-MAPK/ERK-MTOR pathway, which provided a potential target for therapeutic implications. Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; AMPK: adenosine 5’-monophosphate-activated protein kinase; AKT/protein kinase B: AKT serine/threonine kinase; ATG: autophagy related; CDH1/E-cadherin: cadherin 1; CDH2/N-cadherin: cadherin 2; CQ: chloroquine; CTSD: cathepsin D; DIO2: iodothyronine deiodinase 2; DEGs: differentially expressed genes; EECs: endometrial epithelial cells; EMT: epithelial-mesenchymal transition; FN1: fibronectin 1; IUA: intrauterine adhesions; LAMP1: lysosomal associated membrane protein 1; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; T3: triiodothyronine; T4: tetraiodothyronine; TFEB: transcription factor EB; PBS: phosphate-buffered saline; TEM: transmission electron microscopy; TGFB/TGFβ: transforming growth factor beta.

Intrauterine adhesion (IUA) is a common cause of uterine infertility and its histopathologic characteristic is endometrial fibrosis. A shortage of stem cells in the endometrial basalis has been recognized as a common cause of IUA development because approximately 90% of patients suffer from IUA after endometrial injury. In this study, we provide evidence that persistent inflammation is the main contributor to endometrial fibrosis in IUA patients. We further found that treating an IUA-like mouse model with ITI-hUC-MSCs (hUC-MSCs reprogrammed by IL-1β, TNF-α and IFN-γ) significantly decreased endometrial inflammation and fibrosis. Mechanistically, high levels of complement 1 inhibitor (C1INH) secreted by ITI-hUC-MSCs prevented inflammation from inducing profibrotic CD301+ macrophage polarization by downregulating the JAK-STAT signaling pathway. In conclusion, persistent inflammation in the endometria of IUA patients provides macrophage polarization with a profibrotic niche to promote endometrial fibrosis, and the powerful immunomodulatory effects of ITI-hUC-MSCs improve the immune microenvironment of endometrial regeneration.

Macrophages are a key and heterogeneous cell population involved in endometrial repair and regeneration during the menstrual cycle, but their role in the development of intrauterine adhesion (IUA) and sequential endometrial fibrosis remains unclear. Here, we reported that CD301 ⁺ macrophages were significantly increased and showed their most active interaction with profibrotic cells in the endometria of IUA patients compared with the normal endometria by single‐cell RNA sequencing, bulk RNA sequencing, and experimental verification. Increasing CD301 ⁺ macrophages promoted the differentiation of endometrial stromal cells into myofibroblasts and resulted in extracellular matrix accumulation, which destroyed the physiological architecture of endometrial tissue, drove endometrial fibrosis, and ultimately led to female infertility or adverse pregnancy outcomes. Mechanistically, CD301 ⁺ macrophages secreted GAS6 to activate the AXL/NF‐κB pathway, upregulating the profibrotic protein synthesis. Targeted deletion of CD301 ⁺ macrophages or inhibition of AXL by Bemcentinib blunted the pathology and improved the outcomes of pregnancy in mice, supporting the therapeutic potential of targeting CD301 ⁺ macrophages for treating endometrial fibrosis.

Significance: The syndrome of cytotrophoblast invasion beyond the normal boundary (in the superficial myometrium) is collectively termed placenta accreta spectrum. The incidence of this condition is rising. However, little is known about the underlying molecular changes. Global transcriptomic profiling of cytotrophoblasts isolated from these cases, as compared to gestational age-matched controls, revealed numerous changes in gene expression involving diverse pathways, including cell signaling, migration, and immune functions. DOCK4 was the most highly up-regulated mRNA in the cases. Mutations in this gene are mechanistically linked to cancer progression. Overexpression of DOCK4 in primary cytotrophoblasts increased their invasiveness. This study provides molecular insights into the pathways driving placenta accreta spectrum and suggests numerous future directions.

Intrauterine adhesions (IUAs), the leading cause of uterine infertility, are characterized by endometrial fibrosis. The management of IUA is challenging because the pathogenesis of the disease largely unknown. In this study, we demonstrate that the mRNA and protein levels of high mobility group AT-hook 2 (HMGA2) were increased by nearly 3-fold (P < 0.0001) and 5-fold (P = 0.0095) in the endometrial epithelial cells (EECs) of IUA patients (n = 18) compared to controls. In vivo and in vitro models of endometrial fibrosis also confirmed the overexpression of HMGA2 in EECs. In vitro cell experiments indicated that overexpression of HMGA2 promoted the epithelial–mesenchymal transition (EMT) while knockdown of HMGA2 reversed transforming growth factor-β-induced EMT. A dual luciferase assay confirmed let-7d microRNA downregulated HMGA2 and repressed the pro-EMT effect of HMGA2 in vitro and in vivo. Therefore, our data reveal that HMGA2 promotes IUA formation and suggest that let-7d can depress HMGA2 and may be a clinical targeting strategy in IUA.