Breaking the ageing paradigm in endometrium: endometrial gene expression related to cilia and ageing hallmarks in women over 35 years

Does age affect endometrial gene expression?Using unsupervised artificial intelligence methods, we report for the first time that endometrial gene expression changes from 35 years of age in women.Female fertility declines with age, largely attributed to declining oocyte quality and ovarian reserve. Combined with other evidence, a longstanding paradigm holds that age does not affect the endometrial function and age has not been controlled for properly in endometrial studies.A retrospective in silico analysis was performed of endometrial transcriptomic data from the Gene Expression Omnibus (GEO) sample repository for 27 women of different ages. Results were validated in an independent gene expression dataset of 20 endometrial samples from women aged 23-43 years.A systematic search was performed in GEO from October 2016 to January 2019 to identify transcriptomic studies involving women of different ages. Included samples were from norm-ovulatory, women of reproductive age (23-49 years) with regular menstrual cycles who were free of endometriosis and used as controls in a previous endometrial study. We used raw gene expression data and metadata from these samples to investigate the effect of age on endometrial gene expression. Files were downloaded, pre-processed and explored for potential confounding variables and outliers. Artificial intelligence methods were applied to define age groups, and differential expression and functional analyses were applied to demonstrate and understand the effect of age on gene expression at the molecular level. Functional results were validated in an independent gene expression dataset of 20 endometrial samples from women aged 23-43 years.Analysis of the initially retrieved endometrial datasets revealed the age of participants was not available (33.33%) or traceable (43.33%) in most studies. However, one study was suitable for age analysis (GSE4888, n = 27, 23-49 years). Samples showed different transcriptomic profiles according to age, beginning at 35 years. A total of 5778 differentially expressed genes and 27 significantly altered endometrial functions (false discovery rate (FDR) < 0.05) were associated with endometrial gene expression changes related to age. Interestingly, 81.48% of affected functions were related to up-regulation of ciliary processes, with 91 genes involved in cilia motility and ciliogenesis. Other functions included dysregulation of the vascular endothelial growth factor signalling pathway and inhibition of epithelial proliferation triggered by 37 genes involved in cell cycle arrest, angiogenesis, insulin signalling and telomere protection. These findings were validated in an independent dataset using a non-targeted approach; 20 up-regulated ciliary processes (FDR < 0.02) and 6 down-regulated functions related to cell cycle arrest were identified as affected by age, among other hallmarks of ageing such as DNA repair inhibition or sugar metabolism (FDR < 0.05).Data underlying this article are available in GEO, IDs: GSE4888 (main dataset) and GSE102131 (validation dataset).This study is limited in size, as are most studies of endometrial transcriptomics where whole-transcriptome analysis considers nearly 22 000 variables in a relatively small population. Yet, our study includes a main sample set and subsequent validation set that enhances reproducibility of our results and provides reasonable evidence for concluding that age affects endometrial gene expression. A larger study prospectively controlling for patient characteristics is needed to accurately describe changes related to age, with a higher sample size and across a wide age range. Additional studies also are necessary to determine the endometrial ageing contribution to infertility for ultimate translation to a clinical setting.Our findings support an influence of age on the endometrium in a genome-wide functional approach, breaking the endometrial ageing paradigm in human reproduction. To our knowledge, this work is the first to identify, using a genome-wide functional non-targeted approach, ciliary processes as the primary dysregulated function associated with maternal age. These results should guide the research community to control for age as a potential confounding variable in endometrial gene expression studies and to consider endometrial ageing in further studies as a potential cause of infertility in the clinical setting. The reported functional dysregulations could contribute to diminished embryo implantation with age and further studies will demonstrate if such dysregulation underlies some cases of implantation failure. Additionally, the discovery of these functional alterations could enable mechanistic studies, particularly around the age-related increase in uterine pathologies.This research was funded by the Instituto de Salud Carlos III through Miguel Servet programme (CP20/00118) granted to Patricia Diaz-Gimeno (Spanish Government) co-funded by FEDER; and by IVI Foundation (1706-FIVI-041-PD). A.D.-P. (FPU/15/01398) and A.P.-L. (FPU18/01777) are granted by the pre-doctoral programme fellowship from the Ministry of Science, Innovation and Universities (Spanish Government). The authors do not have any competing interests to declare.N/A.