#193 : Comparison of the Different Expression of OCT-4, GATA-3 and Clinical Pregnancy Outcome Between the Day 5 and Day 6 Blastocysts
Authors: Lee Jui-Hua , Chang Wei-Fang , Tzeng Shang-Yu , Lee Yi-Xuan , Tan Shun-Jen , Wang Ruey-Sheng , Ho Jason Yen-Ping , Lin Shyr-Yeu , Hu Yu-Ming , Tzeng Chii-Ruey 
Published: January 2024

Background and Aims: In common experience of in vitro-fertilization (IVF) cycle, blastocysts formed at Day 5 (D5) are regarded as higher quality and are more desirable for embryo transfer compared with those formed at Day 6 (D6). How those delayed D6 embryo differs from D5 embryo is less discussed. This study aimed to elucidate the cell content distinguished by immunofluorescent (IF) staining between the same grade of D5 and D6 blastocysts that might link to the clinical pregnancy outcome in frozen–thawed embryo from different day of blastocyst formation.

Method: The retrospective study included 1327 frozen-thawed single blastocyst transfer (SBT) cycles in the Taipei Fertility Center during March 2020 to December 2022. All D5 (D5-SBT) and D6 (D6-SBT) frozen-thawed SET cycles (n=1327) were separated to non-PGS (n=671) and PGS-euploid (n=656) groups, then assigned to high grade (AA, AB, BA, BB) and low grade (BC, CB, CC). Aneuploidy and mosaicism embryo are excluded. For cell content analysis, pluripotent marker OCT-4 initially expressed in all blastomeres but restricted to the inner cell mass (ICM) of the blastocyst and downregulated in the trophectoderm (TE). GATA-3 is a TE specific marker which increases during pre-implantation embryo development. The cell number, OCT-4 and GATA-3 expression of same grade frozen-thawed D5 (n=14) and D6 (n=14) blastocysts were analyzed with IF staining. D5 extensively cultured to D6 (n=10) were used as the control group of regularly development. Cell numbers of TE and ICM region were examined by OCT-4 and GATA-3 IF detection. The total cell number was visualized with DAPI staining.

Results: According to staining results, there were no significant differences in total cell number, TE and ICM cell number between the same grade D5 and D6 blastocysts. However, TE cell number significantly increased in D5 extensively cultured blastocysts, indicating higher proliferative capacity in TE cells from D5 blastocysts. In the clinical data of the non-PGS group, chemical pregnancy rate (HCG+) and clinical pregnancy rate (SAC+) of high grade D5-SBT were significantly increased compared with high grade D6-SBT (HCG+: 69.1% versus 47.0%, P<0.001; SAC+: 63.0% versus 43.4%, P=0.001). In the PGS-euploid group, the chemical pregnancy rate (HCG+) showed significantly increased in high grade D5-SBT compared with high grade D6-SBT (69.5% versus 60.0%, P=0.047). The clinical pregnancy rate (SAC+) was significantly higher in high grade D5-SBT compared with high grade D6-SBT (65.5% versus 52.5%, P=0.009). The ongoing pregnancy rate was significantly higher in high grade D5-SBT compared with high grade D6-SBT (57.6% versus 43.3%, P=0.005).

Conclusion: According to the IF data in this study, we demonstrated that the number of GATA3-positive TE cells were significantly decreased in D6 blastocyst compared with D5 extensively cultured blastocysts. Due to decreased proliferative capacity in high grade D6 blastocyst, transfer of high grade D5 blastocyst would exhibit higher ongoing pregnancy rate than D6 blastocyst.