Characterization and comparative analyses of transcriptomes for in vivo and in vitro produced peri-implantation conceptuses and endometria from sheep

Abstract

An increasing number of reports indicate that in vitro fertilization (IVF) is highly associated with long‑term side effects on embryonic and postnatal development, and can sometimes result in embryonic implant failure. While high‑throughput gene expression analysis has been used to explore the mechanisms underlying IVF-induced side effects on embryonic development, little is known about the effects of IVF on conceptus-endometrial interactions during the peri-implantation period. Using sheep as a model, we performed a comparative transcriptome analysis between in vivo (IVO; in vivo fertilized followed by further development in the uterus) and in vitro produced (IVP; IVF with further culture in the incubator) conceptuses, and the caruncular and intercaruncular areas of the ovine endometrium. We identified several genes that were differentially expressed between the IVO and IVP groups on day 17, when adhesion between the trophoblast and the uterine luminal epithelium begins in sheep. By performing Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that, in the conceptus, differentially expressed genes (DEGs) were associated mainly with functions relating to cell binding and the cell cycle. In the endometrial caruncular area, DEGs were involved in cell adhesion/migration and apoptosis, and in the intercaruncular area, they were significantly enriched in pathways of signal transduction and transport. Thus, these DEGs are potential candidates for further exploring the mechanism underlying IVF/IVP-induced embryonic implant failure that occurs due to a loss of interaction between the conceptus and endometrium during the peri-implantation period.

Fig. 1.

Summary of experimental design. All ewes were divided randomly into two groups. After either in vivo fertilization and development (IVO control group) or in vitro fertilization and culture (IVP group), at day 6.5, blastocysts were collected and transferred to recipients. At day 17, the recipients in each segment (IVO, n = 37; IVP, n = 20) were sacrificed, and both the conceptuses and the caruncular (C) and intercaruncular (IC) areas of the endometrium with normal morphology were collected from the uterus of recipients (therein defined as the IVO and IVP groups).

Fig. 2.

Number of upregulated and downregulated genes with different fold changes in the conceptus, the caruncular (C) area, and the intercaruncular (IC) area in the IVP group, compared to the IVO group. Positive y-axis (red) represents upregulated gene and negative y-axis (green) represents downregulated gene. Numbers in the bars are upregulated or downregulated genes at each fold change region.

Fig. 3.

Upregulated (green) and downregulated (red) differentially expressed genes based on GO term in the conceptus (P < 0.01). The left ordinate represents the number of DEGs enriched in each term and the right ordinate represents the enrichment score (defined as -Log₁₀ P-value).

Fig. 4.

Upregulated (green) and downregulated (red) differentially expressed genes based on GO term in the caruncular area (P < 0.01). The left ordinate represents the number of DEGs enriched in each term and the right ordinate represents the enrichment score (defined as -Log₁₀ P-value).

Fig. 5.

Upregulated (green) and downregulated (red) differentially expressed genes based on GO term in the intercaruncular area (P < 0.01). The left ordinate represents the number of DEGs enriched in each term and the right ordinate represents the enrichment score (defined as -Log₁₀ P-value).

Fig. 6.

Summary of comparative profiles between the IVP and IVO groups. Observed functional clusters in the present study are highlighted in red text.