Day 3 versus Day 5 refreshment of single-step medium for embryos cultured in a benchtop dry incubator: a randomized sibling oocyte study

Abstract

Research question: Does refreshment of single-step culture medium on Day 3 impact blastocyst formation and ploidy rates in embryos cultured in benchtop dry incubators? Design: This randomized sibling oocyte study was performed between July 2021 and October 2023 including 177 patients who underwent preimplantation genetic testing for aneuploidy (PGT-A). Following intracytoplasmic sperm injection, 3016 metaphase II (MII) oocytes were randomized equally to two culture conditions: (a) medium refreshment on Day 3 (R-D3) or (b) medium refreshment on Day 5 (R-D5). All embryos were cultured in single-step medium in a benchtop dry incubator. No embryo assessments were performed between fertilization check and Day 5. Post-insemination outcomes and ploidy rates were compared between the groups. Results: Fertilization rates were similar between R-D3 and R-D5 (OR 0.89, 95% CI 0.75-1.07; P = 0.212). No significant differences in total usable blastocysts (OR 0.88, 95% CI 0.75-1.02; P = 0.091), euploid blastocysts (OR 0.95, 95% CI 0.80-1.13; P = 0.561) or mosaic blastocysts (OR 1.06, 95% CI 0.72-1.56; P = 0.771) per inseminated MII oocyte were observed. Similar results were observed per two pronuclei (2PN) embryo. However, R-D5 was associated with lower odds of Day 5 blastulation (OR 0.80, 95% CI 0.66-0.98; P = 0.032) and high-quality blastulation per inseminated MII oocyte (OR 0.82, 95% CI 0.68-0.98; P = 0.027), although this was not significant when analysed per 2PN embryo. Sensitivity analyses did not identify interactions between treatment group and cycle characteristics. Conclusion: Omitting the refreshment of single-step medium on Day 3 does not compromise the viability of euploid embryos to blastocyst stage, but does lead to a slight reduction in the number of morphologically high-quality blastocysts.