The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles

Abstract

Purpose: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts?

Methods: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming.

Results: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer.

Conclusion: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

Keywords: Embryo stress response; Mitochondria; RNA-seq; Vitrification-warming; mtDNA content.

Fig. 1

Analysis of mitochondrial DNA (mtDNA) content after blastocyst warming (AW) a) mtDNA content per specimen AW b) mtDNA content per cell observed in aneuploid blastocysts cultured for different amounts of time AW c) mtDNA content per cell measured before vitrification in blastocysts cultured for different periods of time AW d) Average mtDNA content per cell before vitrification (orange dots) and AW (blue triangles) in blastocysts cultured for different time periods AW. * Significant differences (P value < 0.05). Tukey’s multiple comparisons test. Letters above boxes indicate the order from highest to lowest average mtDNA content per cell and whether there are significant differences via post-Anova pairwise tests. If two groups have the same letter above the box, there are no significant differences between them. ** Age in years

Fig. 2

Evaluating RNA-seq data a) Heatmap plot with top 100 most differentially expressed genes in which the two differentiated populations are indicated (red lines) b) Two-dimensional principal component analysis (PCA). Green dots represent blastocysts cultured for 0 h after warming and orange ones represent blastocysts cultured for 4–5 h after warming c) Volcano plots of the study comparison (Blastocysts cultured for 4–5 h after warming (AW) vs blastocysts cultured for 0 h AW) showing the 20 statistically significant DEGs. Red dots show upregulated DEGs and blue dots indicate downregulated DEGs. Genes selected for validation by qRT-PCR are highlighted in blue boxes. *Padj < 0.05 and an absolute log₂ Fold Change of > 2 was considered statistically significant

Fig. 3

Gene set enrichment analysis in blastocysts cultured for 4–5 h after warming (AW) vs blastocysts cultured for 0 h AW. Bars represent normalized enrichment score on Gene Ontology (GO) categories (Padj < 0.05) for top 10 most significant 1) biological processes 2) molecular functions and 3) cellular components of interest in blastocysts cultured 4–5 h vs cultured 0 h and AW. Purple bars indicate upregulation in blastocysts cultured for 4–5 h AW and green bars indicate downregulation in blastocysts cultured for 4–5 h AW

Fig. 4

Enrichment map of the GO biological processes upregulated and downregulated in the study comparison. Processes cluster in different groups. Purple nodes indicate upregulation and green nodes indicate downregulation in blastocysts cultures for 4–5 h after warming. *Padj < 0.05 was considered significantly deregulated