Temporal Trajectory of Plasma miRNA during the Peri-Implantation Stage: Comparing Implantation Success and Failure in Single Frozen-Thawed Blastocyst Transfers

Lu Wang¹Xiang Xiao²Hongya Yang¹Ying Ju¹Xiao He¹Lingyin Kong³Shuqiang Chen¹Xiuyu Feng²Jing Wu¹Yue Liang¹Yuan Ma¹Juan Zhou¹Bo Liang⁴Ni Jin¹Jianlei Huang¹Hai Li¹Xiaohong Wang^1*

• ¹Air Force Medical University Tangdu Hospital, Xi'an, China
• ²Basecare Medical Device Co., Suzhou, China
• ³Jiangsu University, Zhenjiang, China
• ⁴Shanghai Jiao Tong University, Shanghai, China

Background Implantation failure is the most common cause of pregnancy failure and is a major limiting factor in assisted reproduction. Plasma microRNA (miRNA) expression profiles show dynamic changes among individuals with successful implantation. However, the trajectory differences in plasma miRNA expression between women with implantation failure and success, as well as the potential role of those miRNAs are still unclear. Methods This study included 84 women who underwent single frozen-thawed blastocyst transfer in a natural cycle. For each patient, longitudinal plasma samples across five time points throughout the peri-implantation period (Day0/D3/D5/D7/D9) were collected and underwent miRNA sequencing. The failure group (n=27) encountered complete implantation failure, while the success group (n=57) achieved a live birth. Using trajectory analysis and fuzzy c-means clustering, we identified dynamically differentially expressed (DDE) miRNAs and their dynamic expression patterns (DEPs) in a screening set (n=52) and validated findings in an independent validation set (n=32). Clinical correlations, functional annotation, prediction model and in vitro validation of prioritized miRNA-target interactions were systematically conducted. Results 24 DDE miRNAs (FDR<0.05) exhibited five temporal patterns: Recession (R), Growth (G), D3-Trough (T), Multimodal (M), and D5-Trough (T2). The success group predominantly showed M-pattern miRNAs (62.5%), while failures demonstrated pattern transitions (M→T/T2) and exclusive T-pattern expression. Clinical relevance revealed 8 DDE miRNAs associated with at least one clinical variable, with the majority being associated with estradiol/progesterone levels. Functional enrichment implicated Wnt/mTOR pathways in embryo implantation and decidualization. Six DDE miRNAs were successfully replicated in the validation set, while the Support Vector Machines (SVM) model achieved the Area Under the Curve (AUC) of 0.816 (D0) and 0.870 (D3). Additionally, the association between hsa-miR-214-3p and its target gene CTNNB1 was further confirmed in Ishikawa cells. Conclusions Understanding the dynamic landscape changes of the miRNA transcriptome in individuals with implantation failure will help identify dynamic biomarkers from ovulation to the post-implantation stage, providing new insights into the pathological mechanisms of implantation failure and facilitating the research and development of new therapies in the clinical setting.