Unveiling the molecular mechanisms of human platelet lysate in enhancing endometrial receptivity

STUDY QUESTION: Which biological pathways are modulated by primary human endometrial cells in response to in vitro treatment with non-autologous human platelet lysate (HPL)? SUMMARY 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: There is currently no standard therapy for recurrent implantation failure (RIF), and existing treatments have variable effectiveness and do not consistently improve clinical pregnancy rates. Intrauterine infusion of autologous platelet-rich plasma (aPRP), before embryo transfer, promotes endometrial growth and may be the most effective immunomodulatory intervention to significantly improving pregnancy outcomes in RIF patients. HPL is a commercially available, pooled, and cell debris-cleared derivative of PRP suitable for cell culture. STUDY DESIGN, SIZE, DURATION: Cross-sectional (control versus treatment) study including five non-RIF (control) patients and 18 RIF patients. The 18 RIF patients were categorized into two sub-groups: RIF and RIF including thin endometrium (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 h with either serum-free media (SFM) as the untreated control, or SFM supplemented with 1% HPL (EECs), or 10% HPL (ESCs). Cell proliferation was assessed by metabolic assay and immunocytochemistry for Ki-67 expression. Following 48-h 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-h 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-h 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 h significantly increased EEC proliferation by 1.24- to 1.49-fold (P < 0.05) 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 < 0.05). 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 < 0.01) in RIF cultures and a significant 29% increase (from 57.52% to 86.5%, P < 0.01) in RIF+TE cultures. LARGE SCALE DATA: Raw sequencing and count data have been deposited under GEO accession number GSE279514. LIMITATIONS, REASONS FOR CAUTION: One limitation is the small sample size of primary human endometrial samples (N = 23), divided into four patient groups (N = 5-6 per group). Additionally, all participants were pre-menopausal women aged 32-47 years, most of whom fall into the advanced reproductive age category (>35 years), a group often recommended for infertility assessment after 6 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. While research from our group and others suggests that PRP and HPL contain a similar growth factor milieu, randomized controlled trials are necessary to evaluate and compare the efficacy of commercial HPL as a treatment alternative to aPRP. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide the first detailed map of the signaling and extracellular-matrix programs that platelet derivatives activate in primary endometrial cells, offering a mechanistic bridge between the growing clinical use of platelet-rich plasma and its observed improvements in implantation. By clarifying which pathways (chiefly cell-cycle drivers, matrix remodeling enzymes, and intercellular signaling factors) are engaged, the study equips clinicians to refine treatment variables such as dose and timing, and highlights actionable biomarkers that could be monitored to verify a receptive endometrial response. At the scientific level, these insights shift the focus from empirical application to rational modulation of endometrial paracrine signaling, guiding the design of next-generation platelet formulations or synthetic analogs that replicate the same molecular signature with greater consistency and safety. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the CReATe Fertility Centre.

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