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

Does refreshment of single-step culture medium on Day 3 impact blastocyst formation and ploidy rates in embryos cultured in benchtop dry incubators? 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. 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. 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.