Increased OCT4 expression in Day 5 blastocysts may explain the increased clinical outcomes of D5 versus D6 blastocyst transfers

Abstract STUDY QUESTION Are the clinical pregnancy rates and cell lineage-related markers of equal-grade Day 5 (D5) and Day 6 (D6) embryos the same? SUMMARY ANSWER High-grade D5 (HG D5) blastocysts have a higher live birth rate than high-grade D6 (HG D6) blastocysts, which could be associated with differences in intrinsic OCT4 expression. WHAT IS KNOWN ALREADY In IVF cycles, D5 blastocysts are generally considered to be of higher quality and are preferred for embryo transfer over D6 blastocysts. However, the differences between delayed D6 embryos and D5 embryos remain poorly understood. This study aimed to perform an immunofluorescence (IF)-based analysis to evaluate cell content-related differences between same-grade D5 and D6 blastocysts and to assess how these differences may be associated with the clinical pregnancy outcomes following frozen-thawed embryo transfer cycles. STUDY DESIGN, SIZE, DURATION This retrospective study included 774 frozen-thawed single blastocyst transfer (SBT) cycles at the Taipei Fertility Center from March 2020 to February 2023. All D5 (D5-SBT) and D6 (D6-SBT) frozen-thawed cycles (n = 774) were classified as high grade (≥BB, n = 694) or low grade (<BB, n = 80). PARTICIPANTS/MATERIALS, SETTING, METHODS For the cell content analyses, IF staining with the pluripotent marker OCT4 was used to determine the cell numbers in the inner cell mass (ICM), and the trophectoderm (TE) marker GATA3 was used to evaluate the TE region. Same-grade frozen-thawed D5 blastocysts (n = 20), D6 blastocysts (n = 20), and D5 blastocysts cultured to D6 (ExD6, n = 17) were used to assess differences in the cell number in the TE and ICM by evaluating OCT4 and GATA3 expression. The total number of cells was visualized using DAPI staining. OCT4 and GATA3 transcripts were analysed with quantitative reverse transcription polymerase chain reaction (qRT-PCR). MAIN RESULTS AND THE ROLE OF CHANCE For the clinical results, in the single euploid embryo transfer cycle, the overall biochemical pregnancy rate (HCG+, 71.8% vs 52.3%, P < 0.0001), clinical pregnancy rate (SAC+, +66.7% vs 47.7%, P = 0.0001) and live birth rate (59.5% vs 37.9%, P < 0.0001) were significantly higher in the D5-SBT group than in the D6-SBT group. Among HG embryos, the D5-SBT group consistently demonstrated better clinical outcomes. A generalized linear mixed model revealed that the odds ratios were significantly elevated only for D5 embryos but not for other confounders, highlighting the importance of developmental timing. On the other hand, the staining results showed no significant differences in the numbers of TE, ICM, or total cells between the same-grade D5 and D6 blastocysts. However, a notable decrease in OCT4-positive cells was observed in D6 embryos, suggesting a reduction in embryo quality (D5 vs D6: 64 ± 4% vs 49 ± 5% per embryo). ExD6 blastocysts had a significantly increased number of TE cells compared to D6 blastocysts, reflecting greater expansion of the TE population. Gene expression analysis results aligned with these findings, showing a significant decrease in the OCT4/GATA3 ratio in TE cells from D6 embryos compared to D5 embryos LIMITATIONS, REASONS FOR CAUTION This study exclusively analysed euploid embryos in clinical assessments, omitting cases of mosaic embryo transfer. Due to the limited sample size for embryo staining, assessment of additional pluripotent markers was not feasible. WIDER IMPLICATION OF THE FINDINGS Our research demonstrated a significant decrease in the live birth rate with D6 blastocysts compared to D5 blastocysts. The similar proportion of TE cells between D5 and D6 blastocysts suggests that reduced numbers of OCT4-positive cells in D6 blastocysts, rather than TE proliferation, may influence the embryo developmental potential. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was used for this study. W.F.C. is a co-investigator at Taipei Fertility Center and an employee of Taipei Medical Technology Co., Ltd All other authors declare no conflict of interest related to the content of this study. TRIAL REGISTRATION NUMBER N/A.