# Temporal trajectory of plasma miRNA during the peri-implantation stage: comparing implantation success and failure in single frozen–thawed blastocyst transfers

## TL;DR

This study tracks changes in plasma miRNA levels during early pregnancy to understand differences between successful and failed implantations.

## Contribution

The study identifies dynamic miRNA expression patterns and their clinical relevance in implantation success and failure.

## Key findings

- Twenty-four dynamically differentially expressed miRNAs showed five distinct temporal patterns.
- Six miRNAs were validated in an independent set, and an SVM model achieved high AUC values for predicting implantation outcomes.
- The miRNA hsa-miR-214-3p and its target gene CTNNB1 were confirmed to be functionally associated in implantation.

## Abstract

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, remain unclear.

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.

Twenty-four 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 eight 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 machine (SVM) model achieved area under the curve (AUC) values 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.

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.