Screening of Variants in the Transcript Profile of Eutopic Endometrium from Infertile Women with Endometriosis during the Implantation Window / Rastreio de variantes no perfil de tanscritos do endométrio eutópico de mulheres inférteis com endometriose durante a janela de implantação

Abstract Objective Abnormalities in the eutopic endometrium of women with endometriosis may be related to disease-associated infertility. Although previous RNA-sequencing analysis did not show differential expression in endometrial transcripts of endometriosis patients, other molecular alterations could impact protein synthesis and endometrial receptivity. Our aim was to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis and controls during the implantation window. Methods Data from RNA-Sequencing of endometrial biopsies collected during the implantation window from 17 patients (6 infertile women with endometriosis, 6 infertile controls, 5 fertile controls) were analyzed for variant discovery and identification of functional mutations. A targeted study of the alterations found was performed to understand the data into disease's context. Results None of the variants identified was common to other samples within the same group, and no mutation was repeated among patients with endometriosis, infertile and fertile controls. In the endometriosis group, nine predicted deleterious mutations were identified, but only one was previously associated to a clinical condition with no endometrial impact. When crossing the mutated genes with the descriptors endometriosis and/or endometrium, the gene CMKLR1 was associated either with inflammatory response in endometriosis or with endometrial processes for pregnancy establishment. Conclusion Despite no pattern of mutation having been found, we ponder the small sample size and the analysis on RNA-sequencing data. Considering the purpose of the study of screening and the importance of the CMKLR1 gene on endometrial

Resumo Objetivo Anormalidades no endométrio eutópico de mulheres com endometriose podem estar relacionadas à infertilidade associada à doença. Embora a análise prévia de sequenciamento de RNA não tenha evidenciado expressão diferencial em transcritos endometriais de pacientes com endometriose, outras alterações moleculares poderiam afetar a síntese de proteínas e a receptividade endometrial. Nosso objetivo foi rastrear mutações funcionais em transcritos de endométrios eutópicos de mulheres inférteis com endometriose e de controles durante a janela de implantação. Métodos Os dados do sequenciamento de RNA de biópsias endometriais coletados durante a janela de implantação de 17 pacientes (6 mulheres inférteis com endometriose, 6 controles inférteis, 5 controles férteis) foram analisados para a descoberta de variantes e a identificação de mutações funcionais. Um estudo direcionado das alterações encontradas foi realizado para compreender os dados no contexto da doença. Resultados Nenhuma das variantes identificadas foi comuma outras amostras dentro do mesmo grupo, assim como nenhuma mutação se repetiu entre pacientes com endometriose, controles inférteis e férteis. No grupo de endometriose, foram identificadas nove mutações deletérias preditas, mas apenas uma foi previamente associada a uma condição clínica sem impacto endometrial. Ao cruzar os genes mutados com os descritores endometriose e/ou endométrio, o gene CMKLR1 foi associado a resposta inflamatória na endometriose e a processos endometriais para estabelecimento da gravidez. Conclusão Apesar de nenhum padrão de mutação ter sido encontrado, ponderamos o pequeno tamanho da amostra e a análise dos dados de sequenciamento de RNA. Considerando o objetivo do estudo de triagem e a importância do gene CMKLR1 na modulação endometrial, este poderia ser um gene candidato para estudos adicionais que avaliem mutações no endométrio eutópico de pacientes com endometriose.

Is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window?

STUDY QUESTION: Compared to healthy women, is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window (IW)? SUMMARY ANSWER: The eutopic endometrium of infertile women with endometriosis seems to be transcriptionally similar to the endometrium of infertile and fertile controls (FC) during the IW. WHAT IS KNOWN ALREADY: Endometriosis is a disease related to infertility; nevertheless, little is known regarding the ethiopathogenic mechanisms underlying this association. Some studies evaluating the eutopic endometrium of endometriosis patients suggest there is an endometrial factor involved in the disease-related infertility. However, no study to date has evaluated the endometrial transcriptome (mRNA and miRNA) by next generation sequencing (NGS), comparing patients with endometriosis as the exclusive infertility factor (END) to infertile controls (IC; male and/or tubal factor) and FC. STUDY DESIGN, SIZE, DURATION: From November 2011 to November 2015 we performed a case-control study, where 17 endometrial samples (six END, six IC, five FC) were collected during the IW. PARTICIPANTS/MATERIALS, SETTING, METHODS: All endometrial samples had the RNA extracted. Two libraries were prepared for each one (mRNA and miRNA), which were sequenced, respectively, at HISEQ 2500 (RNA-Seq) and MiSeq System (miRNA-Seq), Illumina. The normalization and differential expression were conducted in statistical R environment using DESeq2 package. qPCR was used for data validation, which were analyzed by Kruskal-Wallis test and Dunn posttest (P < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE: RNA-Seq revealed no differentially expressed genes (DEG) among END, IC and FC groups. miRNA-Seq revealed three differentially expressed miRNAs (has-27a-5p, has-miR-150-5p, has-miR-504-5p) in END group compared to FC group. However, none of the miRNAs identified in the sequencing was validated by qPCR. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this study was the small sample size evaluated as a result of the restrictive eligibility criteria adopted, limiting the generalization of the results obtained here. On the other hand, strict eligibility criteria, which eliminated factors potentially related to impaired endometrial receptivity, were required to increase the study's internal validity. WIDER IMPLICATIONS OF THE FINDINGS: This study brings new perspectives on the mechanisms involved in endometriosis-related infertility. The present findings suggest the eutopic endometrium of infertile women with endometriosis, without considering the disease's stage, is transcriptionally similar to controls during the IW, possibly not affecting receptivity. Further studies are needed to evaluate endometrial alterations related to endometriosis' stages. STUDY FUNDING/COMPETING INTEREST(S): This study received financial support from the Sao Paulo Research Foundation (FAPESP-Fundação de Amparo à Pesquisa do Estado de São Paulo; fellowship 2011/17614-6, MGB) and from the National Council for Scientific and Technological Development (CNPq-Conselho Nacional de Desenvolvimento Científico e Tecnológico; INCT-National Institutes of Hormones and Woman's Health, grant 471 943/2012-6, 309 397/2016-2, PAN; fellowship 140 137/2015-7, MGB). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Unremitting cell proliferation in the secretory phase of eutopic endometriosis: involvement of pAkt and pGSK3ß.

OBJECTIVE: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. DESIGN: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated glycogen synthase kinase 3 beta (pGSK3ß), throughout the menstrual cycle. RESULTS: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3ß was identical in all samples and cycle phases, whereas pAkt and pGSK3ß, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. CONCLUSION: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways.

Differential expression of upstream stimulatory factor (USF) 2 variants in eutopic endometria from women with endometriosis: estradiol regulation

BACKGROUND: Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. RESULTS: Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERa) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. CONCLUSION: These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERa and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.