Transcriptome analysis of endometrial tissues following GnRH agonist treatment in a mouse adenomyosis model

Abstract

Purpose: Adenomyosis is a common, benign gynecological condition of the female reproductive tract characterized by heavy menstrual bleeding and dysmenorrhea. Gonadotropin-releasing hormone (GnRH) agonists are one of the medications used in adenomyosis treatment; however, their underlying mechanisms are poorly understood. Moreover, it is difficult to obtain endometrial samples from women undergoing such treatment. To overcome this, we generated an adenomyosis mouse model, which we treated with an GnRH agonist to determine its effect on pregnancy outcomes. We also analyzed endometrial gene expression following GnRH agonist treatment to determine the mechanisms that may affect pregnancy outcome in individuals with adenomyosis.

Methods: Neonatal female mice were divided into a control group, an untreated adenomyosis group, and an adenomyosis group treated with a GnRH agonist (n=6 each). The pregnancy outcome was observed and compared among the groups. Then, three randomly chosen transcriptomes from endometrial tissues from day 4 of pregnancy were analyzed between the adenomyosis group and the GnRH agonist treatment group by RNA sequencing and quantitative reverse transcription polymerase chain reaction (PCR).

Results: The litter size was significantly smaller in the adenomyosis group than in the control group (7±0.28 vs 11±0.26; P<0.05). However, the average live litter size was increased (10±0.28 vs 7±0.28; P<0.05) after GnRH agonist treatment. Three hundred and fifty-nine genes were differentially expressed in the GnRH agonist-treated group compared with the untreated group (218 were downregulated and 141 were upregulated). Differentially expressed genes were related to diverse biological processes, including estrogen metabolism, cell cycle, and metabolite biosynthesis.

Conclusion: GnRH agonist treatment appears to improve the pregnancy outcome of adenomyosis in a mouse model. Besides pituitary down-regulation, other possible mechanisms such as the regulation of cell proliferation may play a role in this. These new insights into GnRH agonist mechanisms will be useful for future adenomyosis treatment.

Keywords: GnRH agonist; RNA-seq; adenomyosis; mouse; pregnancy outcome.

Figure 1

Light microscopy of uteri from the adenomyosis model group and the GnRH agonist treatment group stained with H&E. Notes: A, B: untreated adenomyosis group; C, D: adenomyosis group treated with GnRH agonist (A, C: ×40; B, D: ×100). The arrows in images mean the ectopic endometrial glands. Abbreviations: GnRH, gonadotropin-releasing hormone; H&E, hematoxylin and eosin.

Figure 2

Detection of smooth muscle actin from the adenomyosis model group and the GnRH agonist treatment group by immunohistochemistry. Notes: A, B: untreated adenomyosis group; C, D: adenomyosis group treated with GnRH agonist (A, C: ×40; B, D: ×100). The arrows in images mean the ectopic endometrial glands. Abbreviation: GnRH, gonadotropin-releasing hormone.

Figure 3

Global expression profiles of the adenomyosis group and GnRH agonist-treated group analyzed by cluster analysis. Abbreviation: GnRH, gonadotropin-releasing hormone.

Figure 4

Genes differentially expressed between the adenomyosis group and the GnRH agonist-treated group analyzed by gene ontology and pathways analyses. Abbreviation: GnRH, gonadotropin-releasing hormone.

Figure 5

Ten genes were examined by RT-qPCR analysis based on their fold-change by RNA-Seq and prospective therapeutic value. Note: *P<0.05. Abbreviations: GnRH, gonadotropin-releasing hormone; RT-qPCR, Quantitative reverse transcription polymerase chain reaction.