Endometrial receptivity revisited: endometrial transcriptome adjusted for tissue cellular heterogeneity

STUDY QUESTION Does cellular composition of the endometrial biopsy affect the gene expression profile of endometrial whole-tissue samples? SUMMARY ANSWER The differences in epithelial and stromal cell proportions in endometrial biopsies modify the whole-tissue gene expression profiles and affect the results of differential expression analyses. WHAT IS ALREADY KNOWN Each cell type has its unique gene expression profile. The proportions of epithelial and stromal cells vary in endometrial tissue during the menstrual cycle, along with individual and technical variation due to the method and tools used to obtain the tissue biopsy. STUDY DESIGN, SIZE, DURATION Using cell-population specific transcriptome data and computational deconvolution approach, we estimated the epithelial and stromal cell proportions in whole-tissue biopsies taken during early secretory and mid-secretory phases. The estimated cellular proportions were used as covariates in whole-tissue differential gene expression analysis. Endometrial transcriptomes before and after deconvolution were compared and analysed in biological context. PARTICIPANTS/MATERIAL, SETTING, METHODS Paired early- and mid-secretory endometrial biopsies were obtained from 35 healthy, regularly cycling, fertile volunteers, aged 23–36 years, and analysed by RNA sequencing. Differential gene expression analysis was performed using two approaches. In one of them, computational deconvolution was applied as an intermediate step to adjust for the proportions of epithelial and stromal cells in the endometrial biopsy. The results were then compared to conventional differential expression analysis. Ten paired endometrial samples were analysed with qPCR to validate the results. MAIN RESULTS AND THE ROLE OF CHANCE The estimated average proportions of stromal and epithelial cells in early secretory phase were 65% and 35%, and during mid-secretory phase, 46% and 54%, respectively, correlating well with the results of histological evaluation (r = 0.88, P = 1.1 × 10−6). Endometrial tissue transcriptomic analysis showed that approximately 26% of transcripts (n = 946) differentially expressed in receptive endometrium in cell-type unadjusted analysis also remain differentially expressed after adjustment for biopsy cellular composition. However, the other 74% (n = 2645) become statistically non-significant after adjustment for biopsy cellular composition, underlining the impact of tissue heterogeneity on differential expression analysis. The results suggest new mechanisms involved in endometrial maturation, involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time in context of endometrial receptivity. LARGE-SCALE DATA The RNA-seq data presented in this study is deposited in the Gene Expression Omnibus database with accession number GSE98386. LIMITATIONS REASONS FOR CAUTION Only dominant endometrial cell types were considered in gene expression profile deconvolution; however, other less frequent endometrial cell types also contribute to the whole-tissue gene expression profile. WIDER IMPLICATIONS OF THE FINDINGS The better understanding of molecular processes during transition from pre-receptive to receptive endometrium serves to improve the effectiveness and personalization of assisted reproduction protocols. Biopsy cellular composition should be taken into account in future endometrial ‘omics’ studies, where tissue heterogeneity could potentially influence the results. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by: Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (EU48695); the EU-FP7 Eurostars program (NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (SARM, EU324509); Horizon 2020 innovation program (WIDENLIFE, EU692065); MSCA-RISE-2015 project MOMENDO (No 691058) and the Miguel Servet Program Type I of Instituto de Salud Carlos III (CP13/00038); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526. Authors confirm no competing interests

RNA-Seq statistics of E2, P4, TAM, and RU486 treated and non-treated Ishikawa cells.

*<p>Significant genes (5% FDR) are counted from known genes and compared to non-treated cells.</p>**<p>E2 and P4 significant genes present in human endometrium during the time of embryo implantation.</p

Selection of genes with FPKM >1000 after hormone/modulator treatments.

*<p>FPKM abundance >1000.</p

A Two-Cohort RNA-seq Study Reveals Changes in Endometrial and Blood miRNome in Fertile and Infertile Women

The endometrium undergoes extensive changes to prepare for embryo implantation and microRNAs (miRNAs) have been described as playing a significant role in the regulation of endometrial receptivity. However, there is no consensus about the miRNAs involved in mid-secretory endometrial functions. We analysed the complete endometrial miRNome from early secretory (pre-receptive) and mid-secretory (receptive) phases from fertile women and from patients with recurrent implantation failure (RIF) to reveal differentially expressed (DE) miRNAs in the mid-secretory endometrium. Furthermore, we investigated whether the overall changes during early to mid-secretory phase transition and with RIF condition could be reflected in blood miRNA profiles. In total, 116 endometrial and 114 matched blood samples collected from two different population cohorts were subjected to small RNA sequencing. Among fertile women, 91 DE miRNAs were identified in the mid-secretory vs. early secretory endometrium, while no differences were found in the corresponding blood samples. The comparison of mid-secretory phase samples between fertile and infertile women revealed 21 DE miRNAs from the endometrium and one from blood samples. Among discovered novel miRNAs, chr2_4401 was validated and showed up-regulation in the mid-secretory endometrium. Besides novel findings, we confirmed the involvement of miR-30 and miR-200 family members in mid-secretory endometrial functions.Peer reviewe

Venn diagram showing significant gene expression changes 12 h E2, TAM, P4 or RU486 treatment, relative to non-treated Ishikawa cells.

<p>A Unique and common genes after 12 h E2 and TAM treatment. B Unique and common genes after 12 h P4 and RU486 treatment. The numbers given within each of the circles represent the number of significantly changed genes unique to treatment, and arrows show the manner they are regulated (up- or down-regulation compared to non-treated Ishikawa cells). Overlaps indicate the number of commonly changed genes.</p

Selection of biomarkers related to reproductive system diseases among E2 and P4 significant genes in Ishikawa cell line.

<p>Expression changes are provided in logarithmic scale calculated as following: log (Expression treated/Expression non-treated).</p

Changes in the Transcriptome of the Human Endometrial Ishikawa Cancer Cell Line Induced by Estrogen, Progesterone, Tamoxifen, and Mifepristone (RU486) as Detected by RNA-Sequencing

<div><p>Background</p><p>Estrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively.</p><p>Methodology/Principal findings</p><p>Gene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including <i>PSAP, ATP5G2, ATP5H</i>, and <i>GNB2L1</i> following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: <i>EZH2, MDK, MUC1, SLIT2,</i> and <i>IL6ST</i>, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (<i>CCND1</i>) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as <i>CTNND1, JUP, CDH2, IQGAP1,</i> and <i>COL2A1.</i></p><p>Conclusions</p><p>Significant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage.</p></div

Top 1 network with TAM 12 h significant genes related to DNA replication, recombination and repair, cell cycle, cellular assembly and organization.

<p>Red molecules represent up-regulated and green down-regulated genes among TAM 12 h significant genes in Ishikawa cells. The networks were generated through the use of IPA (Ingenuity® Systems, <a href="http://www.ingenuity.com" target="_blank">www.ingenuity.com</a>).</p

Selection of endometrial specific biomarkers found in 12 h E2 (left) and P4 (right) treated Ishikawa cells and their relative abundance in human endometrial biopsy samples at the time of embryo implantation (n = 2).

<p>Red genes up-regulated in E2 and P4 treated Ishikawa cells compared to non-treated cells; green genes down-regulated. Genes situated on the left side of the diagonal line show higher relative abundance (FPKM) in human endometrial biopsy sample compared to non-treated Ishikawa cells. Genes situated on the right side of the diagonal line show lower relative abundance (FPKM) in human endometrial biopsy sample compared to non-treated Ishikawa cells.</p