Fluo-Cast-Bright: A Deep Learning Pipeline for the Non-Invasive Prediction of Chromatin Structure and Developmental Potential in Live Oocytes

In mammalian oocytes, large-scale chromatin organization regulates transcription, nuclear architecture, and maintenance of chromosome stability in preparation for meiosis onset. Pre-ovulatory oocytes with distinct chromatin configuration exhibit profound differences in metabolic and transcriptional profiles that ultimately determine meiotic competence and developmental potential. Here, we developed a deep learning pipeline for the non-invasive prediction of chromatin structure and developmental potential in live mouse oocytes. Our Fluorescence prediction and Classification on Bright-field (Fluo-Cast-Bright) pipeline achieved 91.3% accuracy in the classification of chromatin state in fixed oocytes and 85.7% accuracy in live oocytes. Importantly, transcriptome analysis following non-invasive selection revealed that meiotically competent oocytes exhibit a higher expression of transcripts associated with RNA and protein nuclear export, maternal mRNA deadenylation, histone modifications, chromatin remodeling and signaling pathways regulating microtubule dynamics during the metaphase-I to metaphase-II transition. Our pipeline provides fast and non-invasive selection of meiotically competent oocytes for downstream research and clinical applications. Methods: Oocyte collection was carried out on non-primed day 20 female mice. Collected cells are maintained in minimal essential medium (MEM) supplemented with 3mg/ml bovine serum albumin (Sigma Aldrich, St. Louis, MO) and milrinone (Sigma; 1 μg/ml) to prevent meiotic resumption. These oocytes are separated into different wells with one oocyte in each well and imaged one by one under the confocal microscope. The 3D bright-field images of each oocyte are then processed by the Fluo-Cast-Bright pipeline and classified into NSN and SN oocytes and prepared for RNA-seq. Allowing for the non-invasive separation of NSN and SN oocytes. A total of 30 oocytes (15 NSN and 15 SN) were sequenced and analyzed in two replicates. Results: Transcriptomics analyses revealed that meiotically competent oocytes exhibit a higher expression of transcripts associated with RNA and protein nuclear export, maternal mRNA deadenylation, histone modifications, chromatin remodeling and signaling pathways regulating microtubule dynamics during the metaphase-I to metaphase-II transition. Our pipeline provides fast and non-invasive selection of meiotically competent oocytes for downstream research and clinical applications.