Vitrification of in Vivo-derived Porcine Morulae and Blastocysts Alters Metabolic and Stress Response Pathway Alterations

Abstract

BackgroundDespite reported promising farrowing rates after non-surgical and surgical transfers of vitrified in vivo-derived porcine morulae and blastocysts (range: 70-75%), their pregnancy loss is yet higher (10-20%) than it is for fresh embryos (< 2.5%). The present study was designed to investigate whether vitrification would affect the transcriptome of porcine morulae and blastocysts, using microarrays and qRT-PCR validation. Material and methods and resultsMorulae and blastocysts were collected from weaned sows (n=13) by laparotomy at Day 6 (Day 0=start of estrus). Of each embryo category, 50 morulae and 50 blastocysts were vitrified (Treatment group) while fresh morulae (n=40) and blastocysts (n=40) acted as control group. After one week of storage, vitrified embryos were cultured in vitrofor 24 h after warming. Non-vitrified morulae (n=40) and blastocysts (n=40), cultured in vitro for 24 h were used as controls. After the in vitro culture period, embryo viability was morphologically assessed. A total of 30 viable embryos per group and embryonic stage(three pools of 10), were subjected to gene expressionanalysis byusing a microarray approach. A fold change cut-off of ±1.5 and a p-unadjusted <0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed morulae and blastocysts were similar to those of the control (nearly 100%, n.s.).A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated) and 105 (112 upregulated and 93 downregulated) in vitrified blastocysts compared to the control group.Transcriptome DEG profiles were dependent on developmental stage, and GO enrichment analysis mainly related DEGs tobiological processes.The vitrification/warming impact on morulae was mostly repression of gene expression with the exception of metabolism-related pathways. In the case of blastocysts, we noted the activation of the cell cycle, cellular senescence andof signaling pathways for TFGβ, p53, FoxO and MAPK. Disruption ofmetabolic-related pathways in morulae and steroid biosynthesis and gap junctions in blastocysts could be related to impaired embryo quality and developmental potential, despite the rather high post-warming survival rates seen in vitro.ConclusionIn conclusion, vitrification modified the transcriptome of in vivo-derived porcine morulae and blastocysts, resulting in moderate gene expression changes, which may disturb subsequent embryo development and pregnancy after embryo transfer.