Inverse embryonic responses of In vivo and In vitro fertilized mouse embryos to vitamin B supplementation during preimplantation period with limited long-term risks.

Vitamin supplementation is commonly used for peri-conceptual consumption, but the long-term impacts are not yet understood. We examined the effects of adding vitamins riboflavin (B2) and pyridoxine (B6) to culture medium on embryo quality produced from in vitro fertilization (IVF) and in vivo fertilization (IVV). At the two-cell stage, IVF and IVV embryos were cultured in a standard medium, Chatot-Ziomel-Bavister (CZB) with (IVVB2B6 and IVFB2B6) or without adding vitamins B2 and B6 (IVVCZB and IVFCZB). For IVVB2B6, vitamin supplementation presented lowered total cell numbers by an elevated inner cell mass (ICM) and reduced trophectoderm (TE) cells (Total cell numbers: IVVCZB = 57 ± 1.6, IVVB2B6 = 44 ± 1.9, IVFCZB = 45 ± 1.6, and IVFB2B6 = 60 ± 4.0; P < 0.0001). In contrast, the IVFB2B6 group showed an inverse response by increased TE cells and reduced placental efficiency after embryo transfer, (fetal:placental ratio: natural mating = 22.7 ± 1.0, IVVCZB = 11.2 ± 0.7, IVVB2B6 = 12.8 ± 1.2, IVFCZB = 12.7 ± 0.5, and IVFB2B6 = 9.5 ± 0.4; P < 0.0002). To investigate the relationship of vitamin B2 and B6 with one-carbon metabolism, by immunostaining heterochromatin methylation levels with H3K27me3. This showed significantly increased intensities in the IVVB2B6 group but not in the IVFB2B6. Oral glucose tolerance test indicated an increased area under the curve (iAUC) in IVVB2B6 at the early adult stage. Our findings suggest that embryo development trajectory with vitamin supplementation is influenced by the method of fertilization, leading to diverse responses.