Project description:Study question : Are there changes in transcript levels as endometrial receptivity changes in patients with recurrent implantation failure (RIF), particularly in alternative splicing events (ASEs), differential expression genes (DEGs) and immune cells levels? Summary answer: Changes in endometrial receptivity affect alternative splicing (AS), differential gene expression and immune cells, and the RNA-binding protein (RBP) KHDRBS3 likelymaybe？ Likely？ is involved in the dysregulation of alternative splicing. What is known already: Endometrial receptivity is critical for RIF patients. Researchers have developed transcript level-based models to predict personalized endometrial assessment at WOI. Investigating alternative splicing dysregulation, immune cells and other transcriptional biomarkers in RIF patients may further help to predict endometrial receptivity and improve embryo implantation success. Study design, size, duration: 90 endometrial samples were collected from fertile healthy controls (HC) during the menstrual cycle, including pre-receptive (PR) (luteinising hormone (LH)+3 days/LH+5 days), receptive (R) (LH+7 days) and post-receptive (PS) (LH+9 days) phases. 73 endometrial samples were taken from RIF patients on hormone replacement therapy (HRT) cycles, including PR (progesterone (P)+3 days), R (P+5 days) and PS (P+7 days) phases. Participants/materials, setting, methods: Total RNA was extracted and sequenced for all 163 endometrial samples . Bioinformatic analysis was used to comprehensively investigate the endometrial transcriptome at different stages of receptivity. We elucidated the biological significance of the universal alteration of alternative splicing in endometrial receptivity and further analyzed the role of immune cells and gene expression in this process. In addition, an ASEs-RBPs correlation network was constructed in the endometrial receptive period to evaluate the dysregulation of AS by RBPs. Main results and the role of chance: Global splicing profiling of the four subgroups (HC: PR vs. R; HC: PS vs. R; RIF: PR vs. R; RIF: PS vs. R) consistently showed that skipped exons (SE) and mutually exclusive exons (MXE) were the most common changes in splicing events. These different ASEs occurred at high frequency and some of them could affect the coding potential of a transcript. A greater number of genes with different alternative splicing events (ASEGs) were identified between PR and R phases than between PS and R phases, and the same trend was observed for differentially expressed genes (DEGs). Both ASEGs and DEGs showed enrichment for pathways associated with cell function and adhesion, while DEGs were also enriched for cytokine and immune-related pathways. Immune cell prediction shows that changes in endometrial receptivity phases result in differences in the levels of CD8 T cells, resting NK cells and other immune cells. During the R phase, there was a significant decrease in CD8 T cells and resting NK cells, whereas monocytes and M0 macrophages were significantly increased compared to the endometrial PR phase in RIF patients. Similarly, analysis of immune cells showed a significant increase in CD8 T cells, M1 and M2 macrophages and a significant decrease in activated NK cells in the R phase compared to the PS phase of the endometrium in RIF patients. Some of these immune cells, such as NK cells and monocytes, are highly correlated with certain ASEs. Investigation of ASEs-RBPs networks suggested that KHDRBS3 may be a key regulator of splicing events. Limitations, reasons for caution: The sample size needs to be increased and cover a variety of situations, such as different age, pathology, dietary habits, etc., and alsothe molecular experimental validation needs to be considered. The lack of direct comparisons between natural and HRT cycles in the same sample and molecular experimental validation need to be considered. Wider implications of the findings: Comprehensive transcriptional analysis of the endometrium provide valuable insight into potential biomarkers related to endometrial receptivity and potential treatments for RIF patients. Study funding/competing interest(s): This study was supported by Supported by the Program of National Natural Science of China (Grant No. 81701409). All authors declared that they have no conflicts of interest.

Project description:Study question: Does a comprehensive study involving two different population cohorts and small RNA sequencing from endometrium and blood reveal new endometrial receptivity-related microRNAs (miRNAs) in healthy conditions and in women with recurrent implantation failure (RIF)? Summary answer: Several annotated miRNAs, not previously associated with endometrial receptivity, as well as one novel miRNA were found as differentially expressed between early secretory vs. mid-secretory phase samples in addition to miRNAs dysregulated in RIF patients. What is known already: The involvement of miRNAs in mid-secretory endometrial functions has been shown and aberrant expression of miRNAs in RIF has been identified in a few previous studies, but not from whole blood samples. However, as the analysis settings and platforms have been different and a small number of samples has been analysed, there is no consensus about the miRNAs involved in endometrial receptivity and dysfunction. Participants/materials, setting, methods: A total of 116 endometrial and 114 blood samples were subjected to Illumina small RNA sequencing. Identification of annotated and novel miRNAs from sequencing reads was performed by miRDeep2 algorithm. miRNAs that had significantly altered levels between the study groups in both EST and ESP datasets were considered as differentially expressed. Ingenuity Pathway Analysis was applied for identifying miRNA target genes and respective canonical pathways from the matched mRNA analysis from the same samples. Custom TaqMan Small RNA assay was used for the validation of novel miRNA (chr2_4401) levels from paired early secretory and mid-secretory endometrial samples. Main results and the role of chance: Among fertile women, 91 differentially expressed miRNAs were identified in mid-secretory vs. early secretory endometrium, while no differentially expressed miRNAs were found in the corresponding blood samples. The comparison of mid-secretory phase samples between fertile women and RIF patients revealed 21 differentially expressed miRNAs from endometrium and one from blood samples. miRNA target gene prediction analysis using our mRNA sequencing data from the matched samples detected several canonical pathways to be involved in mid-secretory endometrial functions, including JAK/STAT signalling, leptin signalling and growth hormone signalling. Moreover, JAK/STAT signalling pathway was also revealed in miRNA target analysis of mid-secretory endometrium from RIF patients when compared to fertile women. Further, our study results highlight the involvement of miR-30 and miR-200 families in endometrial receptivity, and miR-424-5p in the development of endometrial dysfunction. We identified several novel miRNAs, of which miRNA chr2_4401, being 37-fold up-regulated in mid-secretory phase, has a potential role in the mid-secretory endometrial functions. From blood samples we identified miR-30a-5p as a possible marker of infertility related to RIF.

Project description: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: 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%, P<.01) in RIF+TE cultures. 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, most of whom fall into the advanced reproductive age category (>35 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.

Dataset Information

Immune and Transcriptional Landscape of Endometrial Receptivity at P+5 Phase: A Triad Analysis of EIN, RIF, and Control Endometrial Profiles

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