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Nevertheless, the core microorganismal composition in healthy endometrium is not determined and whether the identified bacterial DNA sequences refer to alive\/functionally active microbes is not clear. Furthermore, whether there are cyclical changes in the microbial composition remains an open issue.<\/jats:p><\/jats:sec>STUDY DESIGN, SIZE, DURATION<\/jats:title>RNA sequencing (RNAseq) data from 14 endometrial paired samples from healthy women, 7 samples from the mid-secretory phase and 7 samples from the consecutive proliferative phase were analysed for the microbial RNA sequences.<\/jats:p><\/jats:sec>PARTICIPANTS\/MATERIALS, SETTING, METHODS<\/jats:title>The raw RNAseq data were converted into FASTQ format using SRA Toolkit. The unmapped reads to human sequences were aligned to the reference database Kraken2 and visualised with Krona software. Menstrual phase taxonomic differences were performed by R package metagenomeSeq. The functional analysis of endometrial microbiota was obtained with HUMANn2 and the comparison between menstrual phases was conducted by one-way ANOVA. Human RNAseq analysis was performed using miARma-Seq and the functional enrichment analysis was carried out using gene set enrichment analysis (GSEA; HumanCyc). The integration of metabolic pathways between host and microbes was investigated. The developed method of active microbiota mapping was validated in independent sample set.<\/jats:p><\/jats:sec>MAIN RESULTS AND THE ROLE OF CHANCE<\/jats:title>With the novel metatranscriptomic approach, we mapped the entire alive microbiota composing of &gt;5300 microorganisms within the endometrium of healthy women. Microbes such as bacteria, fungi, viruses and archaea were identified. The validation of three independent endometrial samples from different ethnicity confirmed the findings. Significant differences in the microbial abundances in the mid-secretory vs. proliferative phases were detected with possible metabolic activity in the host-microbiota crosstalk in receptive phase endometrium, specifically in the prostanoid biosynthesis pathway and L-tryptophan metabolism.<\/jats:p><\/jats:sec>LARGE SCALE DATA<\/jats:title>The raw RNAseq data used in the current study are available at GEO GSE86491 and at BioProject PRJNA379542.<\/jats:p><\/jats:sec>LIMITATIONS, REASONS FOR CAUTION<\/jats:title>These pioneering results should be confirmed in a bigger sample size.<\/jats:p><\/jats:sec>WIDER IMPLICATIONS OF THE FINDINGS<\/jats:title>Our study confirms the presence of active microbes, bacteria, fungi, viruses and archaea in the healthy human endometrium with implications in receptive phase endometrial functions, meaning that microbial dysfunction could impair the metabolic pathways important for endometrial receptivity. The results of this study contribute to the better understanding of endometrial microbiota composition in healthy women and its possible role in endometrial functions. In addition, our novel methodological pipeline for analysing alive microbes with transcriptional and metabolic activities could serve to inspire new analysis approaches in reproductive medicine.<\/jats:p><\/jats:sec>STUDY FUNDING\/COMPETING INTERESTS<\/jats:title>This work is supported by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526-R; FEDER\/Junta de Andaluc\u00eda-Consejer\u00eda de Econom\u00eda y Conocimiento: MENDO (B-CTS-500-UGR18) and by the University of Granada Plan Propio de Investigaci\u00f3n 2016 - Excellence actions: Unit of Excellence on Exercise and Health (UCEES) (SOMM17\/6107\/UGR). A.S.-L. and N.M.M. are funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-0854409 and FPU19\/01638). S.A. has received honoraria for lectures from Merck. The funder had no role in this study.<\/jats:p><\/jats:sec>","DOI":"10.1093\/humrep\/deaa372","type":"journal-article","created":{"date-parts":[[2020,12,22]],"date-time":"2020-12-22T12:20:30Z","timestamp":1608639630000},"page":"1021-1031","source":"Crossref","is-referenced-by-count":98,"title":["Mapping the entire functionally active endometrial microbiota"],"prefix":"10.1093","volume":"36","author":[{"given":"Alberto","family":"Sola-Leyva","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada 18071, Spain"},{"name":"Instituto de Investigaci\u00f3n Biosanitaria ibs.GRANADA, Granada 18014, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eduardo","family":"Andr\u00e9s-Le\u00f3n","sequence":"additional","affiliation":[{"name":"Unidad de Bioinform\u00e1tica, Instituto de Parasitolog\u00eda y Biomedicina \u201cL\u00f3pez-Neyra\u201d, CSIC (IPBLN-CSIC), Granada 18016, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nerea M","family":"Molina","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada 18071, Spain"},{"name":"Instituto de Investigaci\u00f3n Biosanitaria ibs.GRANADA, Granada 18014, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura Carmen","family":"Terron-Camero","sequence":"additional","affiliation":[{"name":"Unidad de Bioinform\u00e1tica, Instituto de Parasitolog\u00eda y Biomedicina \u201cL\u00f3pez-Neyra\u201d, CSIC (IPBLN-CSIC), Granada 18016, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julio","family":"Plaza-D\u00edaz","sequence":"additional","affiliation":[{"name":"Instituto de Investigaci\u00f3n Biosanitaria ibs.GRANADA, Granada 18014, Spain"},{"name":"Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Granada 18071, Spain"},{"name":"Children\u2019s Hospital of Eastern Ontario Research Institute, Ottawa, ON K1H 8L1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda Jos\u00e9","family":"S\u00e1ez-Lara","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada 18071, Spain"},{"name":"Instituto de Investigaci\u00f3n Biosanitaria ibs.GRANADA, Granada 18014, Spain"},{"name":"Institute of Nutrition and Food Technology (INYTA), \u201cJos\u00e9 Mataix Verd\u00fa\u201d Biomedical Research Centre (CIBM), University of Granada, Armilla, Granada 18016, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda Carmen","family":"Gonzalvo","sequence":"additional","affiliation":[{"name":"Instituto de Investigaci\u00f3n Biosanitaria ibs.GRANADA, Granada 18014, Spain"},{"name":"Unidad Reproducci\u00f3n, UGC Laboratorio cl\u00ednico y UGC Obstetricia y Ginecolog\u00eda. 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