Dynamic peripheral blood microRNA expression landscape during the peri-implantation stage in women with successful pregnancy achieved by single frozen-thawed blastocyst transfer.

Study question: What are the dynamic expression features of plasma microRNAs (miRNAs) during the peri-implantation period in women with successful pregnancy via single frozen-thawed blastocyst transfer?

Summary answer: There is a significant change in the plasma miRNA expression profile before and after blastocyst transfer, during the window of implantation.

What is known already: The expression of miRNAs in peripheral blood has indicative functions during the peri-implantation period. Nevertheless, the dynamic expression profile of circulating miRNAs during the peri-implantation stage in women with a successful pregnancy has not been studied.

Study design size duration: Seventy-six women treated for infertility with a single frozen-thawed blastocyst transfer in a natural cycle were included in this study. Among them, 57 women had implantation success and a live birth, while 19 patients experienced implantation failure. Peripheral blood samples were collected at five different time points throughout the peri-implantation period, including D0 (ovulation day), D3, D5, D7, and D9 in this cycle of embryo transfer. The plasma miRNAs in women with blastocyst transfer were isolated, sequenced, and analyzed.

Participants/materials setting methods: Peripheral blood samples were collected in EDTA tubes and stored at -80°C until further use. miRNAs were isolated from blood, cDNA libraries were constructed, and the resulting sequences were mapped to the human genome. The plasma miRNAs were initially analyzed in a screening cohort (n = 34) with successful pregnancy. Trajectory analysis, including a global test and pairwise comparisons, was performed to detect dynamic differentially expressed (DE) miRNAs. Fuzzy c-means clustering was conducted for all dynamic DE miRNAs. The correlation between DE miRNAs and clinical characteristics of patients was investigated using a linear mixed model. Target genes of the miRNAs were predicted, and functional annotation analysis was performed. The expression of DE miRNAs was also identified in a validation set consisting of women with successful (n = 23) and unsuccessful (n = 19) pregnancies.

Main results and the role of chance: Following small RNA sequencing, a total of 2656 miRNAs were determined as valid read values. After trajectory analysis, 26 DE miRNAs (false discovery rate < 0.05) were identified by the global test, while pairwise comparisons in addition identified 20 DE miRNAs. A total of seven distinct clusters representing different temporal patterns of miRNA expression were discovered. Nineteen DE miRNAs were further identified to be associated with at least one clinical trait. Endometrium thickness and progesterone level showed a correlation with multiple DE miRNAs (including two of the same miRNAs, hsa-miR-1-3p and hsa-miR-6741-3p). Moreover, the 19 DE miRNAs were predicted to have 403 gene targets, and there were 51 (12.7%) predicted genes likely involved in both decidualization and embryo implantation. Functional annotation for predicted targets of those clinically related DE miRNAs suggested the involvement of vascular endothelial growth factor and Wnt signaling pathways, as well as responses to hormones, immune responses, and cell adhesion-related signaling pathways during the peri-implantation stage.

Large scale data: The raw miRNA sequence data reported in this article have been deposited in the Genome Sequence Archive (GSA-Human: HRA005227) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human/browse/HRA005227.

Limitations reasons for caution: Although the RNA sequencing results revealed the global dynamic changes of miRNA expression, further experiments examining the clinical significance of the identified DE miRNAs in embryo implantation outcome and the relevant regulatory mechanisms involved are warranted.

Wider implications of the findings: Understanding the dynamic landscape of the miRNA transcriptome could shed light on the physiological mechanisms involved from ovulation to the post-implantation stage, as well as identifying biomarkers that characterize stage-related biological process.

Study funding/competing interests: The study was funded by the Major clinical research project of Tangdu Hospital (2021LCYJ004) and the Discipline Platform Improvement Plan of Tangdu Hospital (2020XKPT003). The funders had no influence on the study design, data collection, and analysis, decision to publish, or preparation of the article. There are no conflicts of interest to declare.