FSP1+ stromal cell-associated macrophage efferocytosis contributes to impaired decidualization in adenomyosis

Does disrupted cell crosstalk contribute to embryo implantation failure in adenomyosis, and what is the underlying molecular mechanism?

Expanded Fibroblast-Specific Protein 1 (FSP1)+ stromal cell populations in adenomyosis may promote GAS6-associated macrophage efferocytosis, which correlates with increased macrophage TNF-α secretion and senescence of adjacent stromal cells.

Adenomyosis impairs endometrial receptivity and decidual transformation, contributing to implantation failure and pregnancy loss. Women with this condition exhibit increased endometrial macrophage abundance.

Three mid-secretory phase eutopic endometrial samples from patients with adenomyosis were subjected to single-cell RNA sequencing (scRNA-seq) and integrated with publicly available normal endometrial scRNA-seq data (GSE183837) to characterize cellular alterations and intercellular interactions. Primary human endometrial stromal cells and endometrial macrophages were isolated for in vitro mechanistic studies, including genetic manipulation and functional co-culture assays to investigate stromal-macrophage crosstalk. Peripheral blood mononuclear cells-derived macrophages were generated by M-CSF induction to further validate efferocytosis-related mechanisms, and a mechanical injury-induced C57BL/6 mouse model of adenomyosis was employed to validate key molecular changes in vivo.

ScRNA-seq data were processed with Cell Ranger and Seurat for clustering and differential expression analysis, while cell-cell communication was analyzed using CellPhoneDB and CellChat. Macrophage efferocytosis was assessed by TUNEL/CD68 staining and in vitro co-culture assays. Senescence was evaluated by senescence-associated β-galactosidase staining and senescence-associated markers/senescence-associated secretory phenotype profiling, while decidualization was assessed by mRNA or protein levels of PRL and IGFBP1. Methods for mechanistic studies included adenovirus transduction, quantitative real-time PCR, western blotting, ELISA, chromatin immunoprecipitation-quantitative PCR, immunohistochemistry, immunofluorescence, and EdU staining.

Our findings revealed a significant elevation in the abundance of FSP1+ stromal cells within the eutopic endometrium of adenomyosis patients during the mid-secretory phase. These FSP1+ stromal cells orchestrated GAS6 secretion via the transcription factor ZBTB7A, and GAS6 could potentially interact with MERTK receptors on macrophages to facilitate efferocytosis. Notably, activated macrophages secreted TNF-α, which contributed to senescence in stromal cells and could promote decidualization defects that may further compromise fertility.

The raw scRNA-seq data have been deposited in the NCBI Sequence under accession number PRJNA1270747.

The integration of in-house samples with public data and the relatively small discovery cohort may have introduced bias. The control samples in GSE183837 were obtained from women undergoing IVF for male-factor infertility and may not fully represent unassisted physiological mid-secretory endometrium. Tamnorzatinib is a dual AXL/MERTK inhibitor, so the efferocytosis effects cannot be attributed solely to MERTK signaling. Further studies in larger clinical cohorts, knockout mouse models, and human endometrial assembloids are needed to substantiate the proposed mechanism.

FSP1+ stromal cells play a crucial role in macrophage efferocytosis and decidualization defects. Targeting the GAS6-MERTK-TNF-α pathway may provide a potential therapeutic strategy for treating decidualization impairment in adenomyosis.

This study was supported by the National Natural Science Foundation of China (no. 82401964, 82371678), the Natural Science Foundation of Jiangsu Province (no. BK20240224), and the Noncommunicable Chronic Diseases-National Science and Technology Major Project (no. 2023ZD0518400).

The authors declare no conflict of interest.

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