O-294 Human oocyte survival, early embryo development, metabolic fingerprinting, and pregnancy outcomes following ultra-rapid or standard vitrification and thawing
Authors: Aydin B , Hudkova D , Maggiotto G , Kal N  S , Osmanllari U , Unsal E , Baltaci V , Aktuna S , Yonar D , Dorofeyeva U , Kotliarova E , Vitushynska M , Misyura Y , Yetim I , Babarikova V 
Published: July 2024

Abstract

Study question
Is ultra-rapid vitrification and thawing superior to standard vitrification in terms of preserving oocyte viability, embryo competence and enhancing pregnancy outcomes?

Summary answer
Ultra-rapid vitrification and thawing preserves oocyte viability and embryo quality as confirmed by their euploidy, non-invasive biomarkers in the culture media, and favorable pregnancy outcomes.

What is known already
Cryopreservation and vitrification protocols for oocytes and embryos have varying cooling rates, cryoprotectant chemical composition and concentration. While vitrification avoids intracellular ice crystal formation, the elevated concentrations of cryoprotectants may be cytotoxic. Over the past 20 years, parameters for vitrification protocols were optimized to improve efficiency and viability of human oocytes and embryos, however few studies have evaluated the metabolic and secretome profiles in addition to embryology and pregnancy outcomes, particularly following ultra-rapid vitrification and thawing. Mid-infrared spectroscopy is a great option for characterizing environmental components and metabolic profiles using the more spectrally complex fingerprint region from 1450-500 cm-1.

Study design, size, duration
This prospective study included 1,214 donor oocytes collected between January 2022 and November 2023. Sibling oocytes were assigned to standard or ultra-rapid vitrification and thawing. Corresponding blastocysts underwent the same type of vitrification following trophectoderm biopsies for PGT-A. Spent embryo culture fluid was collected on day 1, 3, and 5 for spectroscopy and PCR analysis, as well as on day 6 for metabolic fingerprinting. Clinical pregnancy was assessed 5 days following single embryo transfer.

Participants/materials, setting, methods
Vitrified oocyte survival was assessed one hour after thawing. Secretome profiling of day 1, 3, and 5 embryos was conducted by Attenuated Total Reflection Fournier Transform Infrared (ATR-FTIR) spectroscopy. Metabolic profiling of blastocysts (day 6) was conducted by Fournier Transform Infrared (FTIR) spectroscopy. Embryology and pregnancy data together with lipid, protein, and nucleic acid expression profiles were evaluated using the Shapiro-Wilk test followed by the student t-test (parametric variables) or the Mann-Whitney U-Test (non-parametric variables).

Main results and the role of chance
There was 100% survival in oocytes subject to ultra-rapid vitrification-thawing (n = 531) compared to 94.5% in oocytes that underwent standard vitrification (n = 634). There were no significant differences in the fertilization rates following intracytoplasmic sperm injection (81.3% with ultra-rapid and 83.2% with standard vitrification) or potential to reach day 3 of embryo development[RL1]. Ultra-rapid vitrification-thawing produced significantly more blastocysts than standard vitrification-thawing (67.3% vs. 53.0%, respectively) but both techniques had similar euploidy rates (61.8% vs. 61.4%, respectively; p &gt; 0.05). Spectra in the 4000-450 cm-1 region revealed there were no significant differences in lipid and protein expression profiles of day 1, 3, 5 and 6 embryos. The similar result obtained for the culture media for day 1,3,5 and 6 day embryo development with no significant difference. Day 3 blastomere biopsy samples and Day5, Day 6 trophectoderm biopsy sample showed no significant differences for total protein concentration, lipid and nucleic acid experssion profiles in both group.This finding was corroborated by PCR analysis showing no significant difference in nucleic acid profiles. Finally, clinical pregnancy rates were 65.22% with ultra-rapid and 56.5% [RL2] with standard vitrification. (p &lt; 0.05)

Limitations, reasons for caution
While both our ultra-rapid and standard vitrification-thawing protocols appear to preserve embryo competence to the blastocyst stage and euploidy, future studies should evaluate whether RNA expression levels are altered with these approaches.

Wider implications of the findings
This study shows competent and euploid embryos can be generated following ultra-rapid vitrification and provides embryo metabolites that can serve as non-invasive biomarkers for the identification of healthy embryos before embryo transfer.

Trial registration number
NA