{"doi":"10.1093/biolre/ioad083","title":"Vitrification preserves follicular transcriptomic dynamics during ex vivo ovulation","abstract":"Dear Editor, Vitrification is an emerging cryopreservation approach for storing cells or tissues at ultra-low temperatures over an extended period of time. Compared with traditional slow-freezing, vitrification is more cost-effective due to the simple and short process. Vitrification is widely used in assisted reproductive technology to cryopreserve gametes, embryos, or gonad tissues for fertility preservation and infertility treatment. We previously demonstrated that a closed vitrification system preserves murine follicle viability, and vitrified follicles exhibited comparable functions to fresh follicles in a 3D encapsulated in vitro follicle growth (eIVFG) system, including follicle maturation, hormone secretion, ovulation, and resumption of oocyte meiosis [1]. Our recent study further demonstrated the faithful preservation of molecular signatures of vitrified murine follicles during follicle-stimulating hormone (FSH)-stimulated maturation [2]. However, it is unknown whether ovulation, another crucial gonadotropin-dependent follicular event, and the involved ovulatory genes and signaling are well conserved in vitrified follicles. Ovulation is a complex dynamic process by which a surge of the luteinizing hormone (LH) from the anterior pituitary stimulates a fully grown preovulatory follicle to release a fertilizable oocyte. LH binds to its surface receptor in mural granulosa cells and theca cells of a preovulatory follicle, and activates a series of signaling molecules to drive key ovulatory events, including follicle rupture, cumulus cell expansion, and resumption of oocyte meiosis I. We have recently demonstrated that the eIVFG-based ex vivo ovulation not only morphologically recapitulates these key ovulatory events but also conserves the ovulatory gene regulatory pathways, such as those related to epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase 1/2 (ERK1/2), progesterone receptor (PGR), inflammation, and proteolysis [3]. Herein, we further determine whether vitrification of immature follicles preserves the ovulation potential at molecular levels when follicles mature to the preovulatory stage in eIVFG. Multilayered secondary follicles isolated from 16-day-old CD-1 mouse ovaries were vitrified as we previously described [1, 2, 4]. With fresh follicles as the control, vitrified follicles were warmed and cultured for 8 days in eIVFG [5, 6]. Vitrified and fresh follicles were grown to preovulatory stage and were freed from alginate encapsulation and treated with 1.5 IU/mL human chorionic gonadotropin (hCG) to induce ovulation ex vivo [3]. Follicles were collected at 0, 1, 4, and 8-h post-hCG for single-follicle ribonucleic acid sequencing (RNA-seq, GEO number: GSE227091) using an optimized SMART-seq2 protocol [7]. Fastq files were generated using bcl2fastq and imported into the Partek Flow software. Reads were aligned to the whole mouse genome assembly-mm10 using the HISAT 2 align, quantified by Ensembl Transcripts release 99 using the Partek EM algorithm, and normalized using the Transcripts Per Million (TPM) method, and differential gene expression analysis was performed using the DESeq2(R). Principal component analysis (PCA) showed distinct follicle separation across post-hCG time points, but separation was not seen between fresh and vitrified follicles at each time (Figure 1A). The volcano plots in Supplemental Figure 1 and Supplemental Table 1 showed all identified differentially expressed genes (DEGs) with fold change >2 or <0.5 and false discovery rate adjusted P-value < 0.05 between vitrified and fresh follicles at each time points. There were 52, 130, 30, and 476 DEGs at 0, 1, 4, and 8-h post-hCG, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis over these DEGs using the functional enrichment tool WebGestalt [8] revealed that none or a few GO terms and signaling pathways were enriched at 0, 1, and 4 h (Supplemental Table 2). At 8 h, DE","journal":"Biology of Reproduction","year":2023,"id":377286,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9586,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":761209,"name":"Daniela D. Russo","orcid":"0000-0002-2244-1080","position":1,"is_corresponding":false},{"id":429965,"name":"Yingzheng Wang","orcid":"0009-0006-1570-3624","position":2,"is_corresponding":false},{"id":395833,"name":"Qiang Zhang","orcid":"0000-0002-8678-7013","position":3,"is_corresponding":false},{"id":372195,"name":"Mary B. Zelinski","orcid":"0000-0002-8513-1549","position":4,"is_corresponding":false},{"id":1413,"name":"Alex K. Shalek","orcid":"0000-0001-5670-8778","position":5,"is_corresponding":false},{"id":88154,"name":"Brittany A. Goods","orcid":"0000-0002-5962-9570","position":6,"is_corresponding":false},{"id":230910,"name":"Shuo Xiao","orcid":"0000-0001-7225-9132","position":7,"is_corresponding":false},{"id":655702,"name":"Jiyang Zhang","orcid":"0000-0003-2099-9512","position":0,"is_corresponding":true}],"reference_count":10,"raw_metadata":null,"created_at":"2026-07-19T01:16:36.370283Z","pmid":"37498173","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}