Transcriptomic profiling of mare endometrium at different stages of endometrosis

Abstract

In the current study, transcriptome profiles of mare endometrium, classified into categories I, IIA, and IIB according to Kenney and Doig, were compared using RNA sequencing, analyzed, and functionally annotated using in silico analysis. In the mild stage (IIA) of endometrosis compared to category I endometrium, differentially expressed genes (DEGs) were annotated to inflammation, abnormal metabolism, wound healing, and quantity of connective tissue. In the moderate stage (IIB) of endometrosis compared to category I endometrium, DEGs were annotated to inflammation, fibrosis, cellular homeostasis, mitochondrial dysfunction, and pregnancy disorders. Ingenuity pathway analysis (IPA) identified cytokines such as transforming growth factor (TGF)-β1, interleukin (IL)-4, IL-13, and IL-17 as upstream regulators of DEGs associated with cellular homeostasis, metabolism, and fibrosis signaling pathways. In vitro studies showed the effect of these cytokines on DEGs such as ADAMTS1, -4, -5, -9, and HK2 in endometrial fibroblasts at different stages of endometrosis. The effect of cytokines on ADAMTS members' gene transcription in fibroblasts differs according to the severity of endometrosis. The identified transcriptomic changes associated with endometrosis suggest that inflammation and metabolic changes are features of mild and moderate stages of endometrosis. The changes of ADAMTS-1, -4, -5, -9, in fibrotic endometrium as well as in endometrial fibroblast in response to TGF-β1, IL-4, IL-13, and IL-17 suggest the important role of these factors in the development of endometrosis.

Figure 1

Shared and unique DEGs in endometria at different stages of endometrosis. Venn diagram showing up-regulated (A) and down-regulated (B) shared and unique DEGs in categories IIA and IIB in comparison to category I endometria in the follicular phase of the estrous cycle.

Figure 2

Endometrial infiltration of macrophages. Representative immunostaining of CD68 in categories I (A) and IIA (B) endometria in the follicular phase of the estrous cycle. (C) negative control; scale bar, 50 µM. The white arrows indicate the macrophages. (D) The number of positive CD68⁺ cells counted in endometrial regions; Asterisks denote statistical differences (*P < 0.05).

Figure 3

Top 10 significant GO biological processes in endometria at different stages of endometrosis. (A) category IIA vs I endometria, (B) category IIB vs I endometria, (C) category IIB vs IIA endometria visualized using ShinyGO software (FDR ≤ 0.05; hypergeometric test followed by FDR correction).

Figure 4

The effect of TGF-β1 on expression of ADAMTS and HK2 mRNA expression in equine endometrial fibroblasts. TGF-β1 treatment (10 ng/ml) on (A–C) ADAMTS1, (D–F) ADAMTS4, (G–I) ADAMTS5, (J–L) ADAMTS9, (M–O) HK2 mRNA transcription in in vitro cultured fibroblast derived from categories I, IIA and IIB endometria. Results are presented as a fold change. Asterisks denote statistical differences (*P < 0.05; **P < 0.01; ***P < 0.001) as determined by the nonparametric Mann–Whitney U test.

Figure 5

The effect of IL-4 on expression of ADAMTS mRNA expression in equine endometrial fibroblasts. IL-4 treatment (10 ng/ml) on (A,B) ADAMTS1, (C,D) ADAMTS4, (E,F) ADAMTS5, (G,H) ADAMTS9 mRNA transcription in in vitro cultured fibroblast derived from (A,C,E,G) categories I and (B,D,F,H) IIA endometria. Results are presented as a fold change. Asterisks denote statistical differences (*P < 0.05; **P < 0.01) as determined by the nonparametric Mann–Whitney U test.

Figure 6

The effect of IL-13 on expression of ADAMTS mRNA expression in equine endometrial fibroblasts. IL-13 treatment (10 ng/ml) on (A,B) ADAMTS1, (C,D) ADAMTS4, (E,F) ADAMTS5, (G,H) ADAMTS9 mRNA transcription in in vitro cultured fibroblast derived from (A,C,E,G) categories I and (B,D,F,H) IIB endometria. Results are presented as a fold change. Asterisks denote statistical differences (**P < 0.01) as determined by the nonparametric Mann–Whitney U test.

Figure 7

The effect of IL-17 on expression of ADAMTS mRNA expression in equine endometrial fibroblasts. IL-17 treatment (10 ng/ml) on (A,B) ADAMTS1, (C,D) ADAMTS4, (E,F) ADAMTS5, (G,H) ADAMTS9 mRNA transcription in in vitro cultured fibroblast derived from (A,C,E,G) categories I and (B,D,F,H) IIB endometria. Results are presented as a fold change. Asterisks denote statistical differences (*P < 0.05) as determined by the nonparametric Mann–Whitney U test.