Here we report that a chemical cocktail (LCDM: hLIF, CHIR99021, DiM and MiH) previously used for extended potential pluripotent stem cells enables the de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs exhibit transcriptomic and epigenetic features characteristic of trophectoderm cells from bovine embryos and retain developmental potency to differentiate into mature trophoblast cells.

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Establishment of Bovine Trophoblast Stem Cells

Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re-expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA-sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos.RNA-sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects.Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos. The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts. Selected validated genes were pointed as potential biomarkers that may help to improve vitrification. 

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Unveiling how vitrification affects the porcine blastocyst: clues from a transcriptomic study

Although vitrification has been widely applied in assisted reproductive technology, it is unknown whether storage time has any impact on the mRNA and lncRNA expression profiles in human embryos. Eleven women (aged 23–35 years) who had undergone in vitro fertilization treatment were recruited for this study. The transcriptomes of 3 fresh eight-cell embryos and 8 surviving vitrified-warmed eight-cell embryos (4 embryos were cryostored for 3 years, and the others were cryostored for 8 years) were analyzed through single-cell RNA-Seq. No differentially expressed mRNAs or lncRNAs were identified between the 3-years group and 8-years group. A total of 128 mRNAs and 365 lncRNAs were differentially expressed in the 8 vitrified-warmed embryos compared with the fresh embryos. The vitrification-warming impact was moderate, and it was mainly related to the pathways of metabolism, stress response, apoptosis, cell cycle, cell adhesion, and signaling for TFG-β and Hippo. The analysis of target mRNAs suggested that lncRNAs might contribute to the regulation of mRNAs after vitrification-warming. Our findings indicated that long-term storage after vitrification does not affect the mRNA and lncRNA expression profiles in human embryos, however, the procedure of vitrification-warming would lead to minor alteration of transcriptome.

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Long-Term Storage Does Not Affect the Expression Profiles of mRNA and Long Non-Coding RNA in Vitrified-Warmed Human Embryos

Effect of bovine oocyte vitrification with EGTA and post-warming recovery with resveratrol on meiotic spindle, mitochondrial function, reactive oxygen species, and developmental competence.

Cryotop vitrification of large batches of pig embryos simultaneously provides excellent postwarming survival rates and minimal interference with gene expression.

Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, invitro-produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n=80) and vitrified (n=80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell-sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation.

Chun Kuen Mak

Papers

Effect of vitrification on global gene expression dynamics of bovine elongating embryos.