Spent Culture Medium miR-372-3p Is Associated with Blastocyst Morphology and Morphokinetic Dynamics in Human Embryos

Background/Objectives: MicroRNAs released into spent culture medium (SCM) may reflect embryo developmental status, but their relationship with blastocyst morphology and time-lapse morphokinetic behavior remains incompletely understood. This prospective study aimed to investigate whether selected SCM microRNAs are associated with blastocyst morphological grade and early morphokinetic dynamics in human embryos. Methods: A total of 432 SCM samples were collected from embryos cultured in a time-lapse incubation system between October 2018 and July 2020. Blastocysts were classified as good, fair, or poor quality according to morphological grading. The expression levels of six candidate microRNAs, including miR-182-5p, miR-302a-3p, miR-372-3p, miR-373-3p, miR-518a-3p, and miR-519d-3p, were quantified using real-time quantitative polymerase chain reaction. Associations between microRNA expression, embryo morphology, and morphokinetic parameters were analyzed using GEE, multivariate logistic regression, and receiver operating characteristic (ROC) analysis. Results: After accounting for within-patient clustering using GEE, good-quality embryos showed significantly lower expression of miR-372-3p and miR-373-3p than poor-quality embryos. On multivariate logistic regression, lower miR-372-3p expression remained independently associated with optimal t5-t2 interval after adjustment, whereas its associations with optimal CC3 and the combined outcomes were attenuated. ROC analysis demonstrated modest discriminatory ability for miR-372-3p alone (AUC = 0.644), whereas embryo morphology alone achieved an AUC of 0.731, and the combined model incorporating morphology and miR-372-3p reached the highest AUC of 0.752. Conclusions: These findings suggest that SCM microRNA expression is associated with embryo development. In particular, reduced miR-372-3p expression may reflect more favorable blastocyst morphology and early developmental dynamics, supporting its potential as an adjunctive molecular indicator of embryo quality. Further validation incorporating embryo ploidy status and clinical outcomes is warranted before clinical application.