Effect of multiple freezes and thaws on FET success following biopsy and PGS

Abstract

sperm collected from cauda epididymis (C57BL6 inbred and B6D2F1 outbred) for 4-6h. Zygotes were cultured either in suboptimal Whitten's medium and 20% O2 (IVFWM) or in optimal KSOM medium with amino acids and 5% O2 (IVFKAA) for 96h in 37 C. Control blastocysts were flushed from the uterus of mated mice (FB mice). Blastocysts were transferred to pseudo-pregnant recipients. Pups were reared to adulthood and peripheral tissues were collected and serum corticosterone levels measured. Total RNA and protein were isolated from fat, muscle and liver. mRNA expression of GR and a selected group of GR-downstream target genes were analyzed by qPCR; GR protein level by Western Blot. One-way AN-OVA with post hoc correction was used as appropriate; p<0.05 was considered significant.RESULTS: Serum corticosterone levels were not different. Increased GR expression was observed in blastocysts conceived by IVF, being higher in the group using suboptimal conditions (IVFWM) in both strains. Interestingly, fat tissue of both inbred and outbred males exhibited a 2-fold increase in the expression of GR mRNA and protein. Instead, females did not exhibit any changes in GR levels in all the tissues tested. Finally, known downstream targets of GR were upregulated in the fat tissue of male mice.CONCLUSIONS: Male mice conceived by IVF have higher GR levels and GR-target genes expression in fat tissues. Importantly, these findings indicate that memory of the preimplantation environment is maintained in adulthood in a tissue and sex specific manner. The metabolic implications of these findings are discussed. Concurrent studies are further evaluating epigenetic changes at the GR promoter.