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fertility centrehttps://www.ebi.ac.uk/ena/browser/view/PRJNA1173173Homo sapiensxref:PubMed:40663777Summary Answer: HPL treatment stimulates endometrial growth and trophoblast attachment by activating cell proliferation, and modulating cell-cell signaling and extracellular matrix organization. What is known already: Despite advancements in RIF management, there is currently no standard therapy, and existing treatments have variable effectiveness and do not consistently improve clinical pregnancy rates. There is a general consensus that intrauterine infusion of aPRP, before embryo transfer, promotes endometrial growth and may be the most effective immunomodulatory intervention to significantly improving pregnancy outcomes in RIF patients. Study design, size, duration: Cross-sectional (control versus treatment) study including five non-RIF (control) patients and eighteen RIF patients from the CReATe Fertility Centre, Toronto. The eighteen RIF patients were categorized into two sub-groups: RIF and RIF+TE. Participants/materials, setting, methods: Endometrial tissue was collected from pre-menopausal women (32-47 years of age) during routine biopsy procedures at the CReATe Fertility Centre, Toronto. Primary endometrial epithelial (EECs) and stromal cells (ESCs) were enzymatically isolated, cultured separately, and treated for 48 hours with either SFM (SFM) as the untreated control, or SFM supplemented with 1% HPL (EECs), or 10% HPL (ESCs). Cell proliferation was assessed using the PrestoBlueTM reagent (metabolic assay) and immunocytochemistry for Ki-67 expression. Following 48-hour treatment, total RNA was isolated from untreated and treated cells to prepare pooled RNA libraries, which were then subjected to RNA sequencing (150 cycles paired-end). Differential gene expression was performed using the DESeq2 package and RStudio/R. Significant differentially expressed genes were determined with the following cut off values: log2FoldChange >|2| and padj <0.05. Pathway enrichment analysis was then performed with Enrichr (Reactome 2022 database) to identify enriched pathways. After 48-hour treatment with SFM or HPL, a trophoblast attachment assay was also performed with fluorescently labeled HTR-8/SVneo trophoblast spheroids, where spheroids were seeded on top of pre-treated EEC monolayers for a 1-hour incubation to allow for attachment. Fluorescent microscopy and ImageJ™ software were used to image and quantify the total number of seeded and attached spheroids. Main results and the role of chance: Treatment with non-autologous HPL for 48 hours significantly increased EEC proliferation by 1.24- to 1.49-fold (P<.05) in all groups. ESCs showed a significant proliferation increase of 1.29-fold in the proliferative phase RIF group and 1.92-fold in the secretory phase RIF+TE group (P<.05). HPL treatment upregulated 45 genes in EECs, including MMP1, MMP9, and ADAMTS18, while 378 genes were upregulated in ESCs, such as BUB1, CDK1, MKI67, and PLK1. Twenty-two common genes were significantly upregulated in both cell types. EECs had 30 downregulated genes, including KL and ADRA2A, while ESCs had 429 downregulated genes, such as PTGIS, PTGDS, and PTGES, with seven common genes downregulated in both cell types. Pathway enrichment analysis revealed that upregulated pathways in EECs included extracellular matrix organization and degradation, while ESCs showed enrichment in cell cycle (mitotic), cell cycle checkpoints, and extracellular matrix degradation. Downregulated pathways included receptor signaling of the fibroblast growth factor receptor 1 in EECs, prostaglandin synthesis in ESCs, and G-protein coupled receptor signaling in both cell types. HPL treatment also increased primary EEC attachment to trophoblast spheroids compared to the untreated control. This increased attachment was consistent in EECs from RIF patients, regardless of endometrial thickness, with a 26% increase (from 42.58% to 68.90%, P<.01) in RIF cultures and a significant 29% increase (from 57.52% to 86.5%, P35 years), a group often recommended for infertility assessment after six months of unsuccessful conception attempts. Although our study utilized primary endometrial cells and indicates that HPL may be an effective treatment for RIF and TE, these in vitro findings need to be validated in vivo. Clinical studies, such as randomized controlled trials, are necessary to evaluate and compare the efficacy of commercial HPL as a treatment alternative to aPRP. These trials would also allow for testing the safety of using a commercial non-autologous HPL product in human patients and help optimize treatment protocols, including the ideal dosage, concentration, timing, and treatment duration to maximize therapeutic benefits and develop more effective treatments for patients with endometrial causes of RIF. Wider implications of the findings: Platelet-rich plasma (PRP) treatment for RIF and TE is increasingly being adopted by fertility clinics worldwide. Our research represents the first detailed investigation into the molecular effects of HPL, a derivative of PRP, on primary endometrial cells from patients with RIF, establishing a foundation for understanding how PRP/HPL impacts endometrial growth and receptivity. Our study demonstrated that commercial non-autologous HPL stimulates endometrial cell proliferation, modulates the expression of factors related to cell mitosis, extracellular matrix remodeling, and cell-cell signaling, and enhances endometrial-embryo attachment. By elucidating the mechanistic actions of HPL, our research provides valuable insights into this therapeutic approach and informs future treatments aimed at improving embryo transfer success. While these findings suggest that HPL could be as effective as aPRP, further investigation is necessary to evaluate its safety and efficacy in humans, necessitating comprehensive studies before HPL can be routinely used in clinical practice. Study funding/competing interest(s): This study was funded by the CReATe Fertility Centre. Overall design: Human primary endometrial cells, stromal or epithelial. Cells originated from patients with RIF with or without thin endometria, from the secretory or proliferative phases. Control samples from patients with successful pregnancies were also included. Cells were either at P0 immediately post collection, or at P1 with or without treatment of HPL.ENAfalseUnveiling the Molecular Mechanisms of HPL in Enhancing Endometrial ReceptivityUnveiling the Molecular Mechanisms of HPL in Enhancing Endometrial Receptivity2025-09-242025-05-05PRJNA1173173GSE279514960640663777