HSP70-Mediated Macrophage-Stromal Crosstalk is an Immunopathogenic Driver of Recurrent Implantation Failure

Recurrent implantation failure (RIF) remains a major challenge in assisted reproductive technology (ART), affecting a considerable proportion of patients despite multiple transfers of high-quality embryos. Although impaired endometrial receptivity is recognized as a critical contributor to RIF, the molecular mechanisms responsible for establishing a non-receptive endometrial microenvironment remain incompletely understood. The proliferative phase precedes decidualization and subsequent embryo implantation, representing a critical period for endometrial preparation. The endometrium is a highly dynamic tissue composed of diverse epithelial, stromal, vascular, and immune cell populations that undergo coordinated remodeling throughout the menstrual cycle; however, the cell type-specific alterations and pathogenic immune-stromal interactions during this phase have not been comprehensively characterized.

This study aimed to dissect the transcriptional landscape of the proliferative-phase endometrium in RIF patients, and validate the key pathways through experimental approaches.

We for the first time performed single-cell RNA sequencing (scRNA-seq) of proliferative-phase endometrial tissues from RIF patients and control subjects to characterize cell-type-specific transcriptional alterations and intercellular communication patterns. Peripheral blood scRNA-seq was additionally performed to determine whether immune alterations were tissue-specific or systemic. The impact of HSP70 on the transcriptome of endometrial stromal cells was investigated using RNA sequencing. The production of HSP70 and CCL7 in RIF patients was measured by enzyme-linked immunosorbent assay (ELISA). The HSP70/CCL7 axis-mediated functional crosstalk between macrophages and endometrial stromal cells was investigated using Transwell assay, QPCR, and SA-β-Gal staining. HSP70-induced activation of PI3K signaling was predicted using RNA sequencing data, then validated through Western blot, ELISA, and Transwell assay.

We identified excessive inflammatory immune infiltration as a microenvironmental hallmark of RIF endometrium, as reflected by increased percentages of T cells, monocytes, and NK cells. This alteration is associated with pathogenic macrophage - stromal crosstalk. Mechanistically, an inflammatory milieu promotes the expression of 70-kDa heat shock protein (HSP70) in macrophages, which stimulates CCL7 production in endometrial stromal cells via PI3K signaling. CCL7 drives the recruitment of multiple types of immune cells that further amplify the inflammation. Neutralization of CCL7 or inhibition of PI3K signaling prevented this pro-inflammatory infiltration. The HSP70-CCL7 axis facilitates endometrial stromal cell senescence and leads to impaired decidualization.

This study provides the first single-cell transcriptomic atlas of proliferative-phase endometrium in RIF and identifies a pathogenic macrophage-stromal communication network associated with HSP70/CCL7 axis. Our findings reveal that inflammatory microenvironmental remodeling occurs before the window of implantation and contributes to subsequent endometrial dysfunction. Targeting HSP70/CCL7-mediated immune-stromal crosstalk may represent a promising therapeutic strategy for early intervention and prevention of RIF.