Aging promotes accumulation of senescent and multiciliated cells in human endometrial epithelium.

Study question: What changes occur in the endometrium during aging, and do they impact fertility?

Summary answer: Both the transcriptome and cellular composition of endometrial samples from women of advanced maternal age (AMA) are significantly different from that of samples from young women, suggesting specific changes in epithelial cells that may affect endometrial receptivity.

What is known already: Aging is associated with the accumulation of senescent cells in aging tissues. Reproductive aging is mostly attributed to the decline in ovarian reserve and oocyte quality, whereas the endometrium is a unique complex tissue that is monthly renewed under hormonal regulation. Several clinical studies have reported lower implantation and pregnancy rates in oocyte recipients of AMA during IVF. Molecular studies have indicated the presence of specific mutations within the epithelial cells of AMA endometrium, along with altered gene expression of bulk endometrial tissue.

Study design size duration: Endometrial transcriptome profiling was performed for 44 women undergoing HRT during the assessment of endometrial receptivity before IVF. Patients younger than 28 years were considered as the young maternal age (YMA) group (age 23-27 years) and women older than 45 years were considered as the AMA group (age 47-50 years). Endometrial biopsies were obtained on Day 5 of progesterone treatment and RNA was extracted. All endometrial samples were evaluated as being receptive based on the expression of 68 common endometrial receptivity markers. Endometrial samples from another 24 women classified into four age groups (YMA, intermediate age group 1 (IMA1, age 29-35), intermediate age group 2 (IMA2, age 36-44), and AMA) were obtained in the mid-secretory stage of a natural cycle (NC) and used for validation studies across the reproductive lifespan.

Participants/materials setting methods: A total of 24 HRT samples (12 YMA and 12 AMA) were subject to RNA sequencing (RNA-seq) and differential gene expression analysis, 20 samples (10 YMA and 10 AMA) were used for qPCR validation, and 24 NC samples (6 YMA, 6 IMA1, 6 IMA2 and 6AMA) were used for RNA-seq validation of AMA genes across the woman's reproductive lifespan. Immunohistochemistry (IHC) was used to confirm some expression changes at the protein level. Computational deconvolution using six endometrial cell type-specific transcriptomic profiles was conducted to compare the cellular composition between the groups.

Main results and the role of chance: Comparisons between YMA and AMA samples identified a lower proportion of receptive endometria in the AMA group (P = 0.007). Gene expression profiling identified 491 differentially expressed age-sensitive genes (P adj < 0.05) that revealed the effects of age on endometrial epithelial growth and receptivity, likely contributing to decreased reproductive performance. Our results indicate that changes in the expression of the cellular senescence marker p16^INK4a and genes associated with metabolism, inflammation, and hormone response are involved in endometrial aging. Importantly, we demonstrate that the proportion of multi-ciliated cells, as discovered based on RNA-seq data deconvolution and tissue IHC results, is affected by endometrial aging, and propose a putative onset of age-related changes. Furthermore, we propose that aging has an impact on the transcriptomic profile of endometrial tissue in the context of endometrial receptivity.

Large scale data: The raw sequencing data reported in this article are deposited at the Gene Expression Omnibus under accession code GSE236128.

Limitations reasons for caution: This retrospective study identified changes in the endometrium of patients undergoing hormonal replacement and validated these changes using samples obtained during a NC. However, future studies must clarify the importance of these findings on the clinical outcomes of assisted reproduction.

Wider implications of the findings: The findings reported in this study have important implications for devising future strategies aimed at improving fertility management in women of advanced reproductive age.

Study funding/competing interests: This research was funded by the Estonian Research Council (grant no. PRG1076), Horizon 2020 innovation grant (ERIN, grant no. EU952516), Enterprise Estonia (grant no. EU48695), MSCA-RISE-2020 project TRENDO (grant no. 101008193), EU 874867 project HUTER, the Horizon Europe NESTOR grant (grant no. 101120075) of the European Commission, the EVA specialty program (grant no. KP111513) of the Maastricht University Medical Center (MUMC+), MICIU/AEI/10.13039/501100011033 and FEDER, EU projects Endo-Map (grant no. PID2021-12728OB-100), ROSY (grant no. CNS2022-135999), and the National Science Fund of Bulgaria (grant no. KII-06 H31/2). The authors declare no competing interests.