Ultra-rapid versus conventional oocyte vitrification: a comparative study of clinical and molecular outcomes with...

By reducing the duration of the vitrification-warming procedure to 1 min does ultra-rapid vitrification-warming (URV/W) effectively maintain oocyte viability and improve clinical outcomes compared to conventional vitrification-warming (CV/W) timing (14 min) and what is the effect of the two procedures on cellular stress?

URV/W shortens procedure time, reduces cryoprotectant exposure, improves oocyte survival, and is associated with fewer transcriptomic alterations in oocytes than CV/W.

CV/W techniques require extended cryoprotectant exposure and micromanipulation, which elevate cellular stress and the risk of cryoinjury. URV/W procedures address these challenges by reducing exposure and manipulation, though comparative data on clinical outcomes and transcriptomic signatures in human oocytes remain limited.

The study was performed between August 2024 and December 2024 and included a clinical cohort and a transcriptomic cohort to provide a thorough assessment of the effects of cryopreservation methodology on oocyte and embryo viability, development, and cellular stress. The clinical cohort comprised 1077 oocytes used to evaluate clinical outcomes following CV/W and URV/W. The study also included a prospective analysis involving 68 oocytes from 4 donors in the transcriptomic cohort. Oocytes and trophectoderm biopsy samples from blastocyst-stage embryos were assigned to three groups: fresh, CV/W, and URV/W.

The clinical study was performed using a total of 1077 oocytes from 46 donors, which were matched and allocated for the comparison of clinical outcomes between CV/W (n = 519) and URV/W (n = 558). Following vitrification-warming, the oocytes were fertilized via ICSI for assessment of embryo development and clinical outcomes. In the transcriptomic cohort of the study, transcriptomic testing and analysis were performed as part of the prospective analysis to compare CV/W and URV/W in terms of their effectiveness in oocyte freezing and warming, their impact on embryo development, and their effects at the molecular level. A total of 68 samples were obtained from 4 donors, including eight oocytes and eight trophectoderm biopsy samples from each of the three groups. These samples were analyzed to investigate the cellular effects of cryopreservation-induced stress through transcriptomic profiling. The single-cell transcriptomic study included preparation of cDNA libraries followed by next-generation sequencing to investigate differential gene expression across oocytes and embryonic trophectoderm cells in the three groups. This method allowed for the comprehensive monitoring of transcriptional activity, enabling the detection and quantification of mRNA molecules to evaluate the transcriptomic signatures of the study groups. In addition, functional enrichment analyses of up- and downregulated genes were conducted and classified based on gene ontology to identify potential pathways associated with embryo quality and cellular stress.

Transcriptomic analysis indicated that gene expression patterns following URV/W were more similar to fresh oocytes than those undergoing CV/W. Differences in gene expression patterns among blastocysts from the three groups were minimal, as the total number of differentially expressed genes was limited. Oocytes subjected to URV/W demonstrated a significantly higher survival rate, lower post-ICSI degeneration, and produced more high-quality Day-5 blastocysts compared to those vitrified using CV/W methods (P < 0.001 for all comparisons). These findings are unlikely to be solely attributable to differences in procedural timing, as fertilization, cleavage, euploidy, clinical pregnancy, and live birth rates were comparable between groups (P > 0.05 for all comparisons).

All oocyte samples donated (1145) for research purposes were included in the study without quality-based selection. Only embryos reaching the blastocyst stage on Days 5 or 6 using the URV/W method were included in the transcriptomic analysis. Embryos not reaching these stages were excluded. Consequently, only data from embryos with optimal development and good quality were analyzed, and compared among the three groups. Obtaining cDNA in single-cell studies is a novel and challenging process, particularly due to the limited availability of RNA. While advanced clinical evaluations can still be conducted on biopsied oocytes and embryos, the failure to obtain cDNA from the same samples may result in incomplete data, which can limit the overall scope and outcomes of the study.

The comprehensive single-cell transcriptomic data obtained from this expanded dataset will help delineate pathways altered by different vitrification methods, providing critical insights into their efficacy and potential risks in clinical practice. Additionally, the findings will illuminate key genes and pathways involved in embryonic stress, enabling the development of more cost-effective, targeted gene panels for evaluating these factors.

This study was funded by the Sanatórium pre liečbu neplodnosti SPLN's, Ovogene and Mikrogen Genetic Diagnosis Laboratory's own resources. A.V.P. is a Medical Science Liaison for Kitazato Corporation-Dibimed. His role in the study was strictly scientific and unrelated to any commercial interest. All the other co-authors declare no conflicts of interest.

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