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We thank all men who generously donated testicular material for the purpose of this study. We also acknowledge the research support by Copan Italia S.p.A Inc., and Clearblue, SPD Swiss Precision Diagnostics GmbH. This study is part of a PhD Thesis conducted at the Official Doctoral Program in Biomedicine of the University of Granada, Spain. This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grant numbers RYC-2016-21199 and ENDORE (SAF2017-87526-R); by FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento: MENDO (B-CTS-500-UGR18); by Junta de Andalucia: (PAIDI P20_00158) by the University of Granada, Plan Propio de Investigacion 2016, Excellence actions: Units of Excellence; Unit of Excellence on Exercise and Health (UCEES), and the Junta de Andalucia, Consejeria de Conocimiento, Investigacion y Universidades and European Regional Development Fund: (SOMM17/6107/UGR); by Spanish Ministry of Science, Innovation, and Universities: (PRE2018085440 and FPU19/01638); and by Spanish Ministry of Education, Culture, and Sport: (FPU15/01193). Funding for open access charge: Universidad de Granada/CBUA Sequence data of all testicular spermatozoa and negative control samples have been deposited in the National Centre for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/sra) under the BioProject ID PRJNA643898. The preliminary results of this study were presented as a poster communication at the 35th Annual ESHRE Meeting (Vienna, 2019)., Research question: The semen harbours a diverse range of microorganisms. The origin of the seminal microbes, however, has not yet been established. Do testicular spermatozoa harbour microbes and could they potentially contribute to the seminal microbiome composition? Design: The study included 24 samples, comprising a total of 307 testicular maturing spermatozoa. A high-throughput sequencing method targeting V3 and V4 regions of 16S rRNA gene was applied. A series of negative controls together with stringent in-silico decontamination methods were analysed. Results: Between 50 and 70% of all the detected bacterial reads accounted for contamination in the testicular sperm samples. After stringent decontamination, Blautia (P = 0.04), Cellulosibacter (P = 0.02), Clostridium XIVa (P = 0.01), Clostridium XIVb (P = 0.04), Clostridium XVIII (P = 0.02), Collinsella (P = 0.005), Prevotella (P = 0.04), Prolixibacter (P = 0.02), Robinsoniella (P = 0.04), and Wandonia (P = 0.04) genera demonstrated statistically significant abundance among immature spermatozoa. Conclusions: Our results indicate that the human testicle harbours potential bacterial signature, though in a low-biomass, and could contribute to the seminal microbiome composition. Further, applying stringent decontamination methods is crucial for analysing microbiome in low-biomass site., Copan Italia S.p.A Inc., Clearblue, SPD Swiss Precision Diagnostics GmbH, Spanish Government, European Commission RYC-2016-21199 SAF2017-87526-R, FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento: MENDO B-CTS-500-UGR18 Junta de Andalucia PAIDI P20_00158, University of Granada, Plan Propio de Investigacion 2016, Excellence actions: Units of Excellence, Unit of Excellence on Exercise and Health (UCEES), Junta de Andalucia Consejeria de Conocimiento, Investigacion y Universidades, European Commission SOMM17/6107/UGR, Spanish Government PRE2018085440 FPU19/01638 FPU15/01193, Universidad de Granada/CBUA Sequence BioProject PRJNA643898

Study question What is the entire metabolomic profile of human semen and does the metabolic composition differ between men with good-quality and low-quality semen? Summary answer Human semen contains ∼700 different metabolites, and the metabolomic signature differs between normozoospermic men and men with altered seminal parameters. What is known already Semen contains a wide diversity of metabolites as has been identified in single and targeted metabolite studies. The full composition of metabolites in human semen, however, is not known. The knowledge of the complete metabolic signature in semen and whether there are differences between metabolic composition and seminal quality could enhance our knowledge of possible factors involved in reduced sperm quality and male infertility. Study design, size, duration Case-control study, where a total of 100 men (age= 29.73±8.9 years) from March 2019 to March 2020 participated. The study was approved by the Ethics Committee of Investigación Biomédica de Andalucia. Participants/materials, setting, methods Semen samples from 69 normozoospermic and 31 oligozoospermic men were collected at the University Hospital and sperm biobank (Ceifer Biobank - NextClinics). The complete metabolome from unprocessed seminal samples was analysed by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS). Raw data were extracted, peak-identified and quality control processed using Metabolon’s hardware and software (metabolon.com). Multiple regression models controlling for age and sample collection centres were applied using R software. Main results and the role of chance In total, 695 different metabolites were detected in the seminal samples, where docosahexaenoate (DHA; 22:6n3, PubChem ID 445580), choline phosphate (1014), dihomo-linolenate (20:3n3 or n6, 5280581), docosapentaenoate (n6 DPA; 22:5n6, 6441454), adenosine 3’,5’-cyclic monophosphate (cAMP, 6076) and N-acetylalliin (122164824) metabolites were the most prevalent. The seminal metabolomic profiles differed significantly between men with normal and low sperm parameters. The most abundant metabolites in normozoospermic men belonged to Lipid Super-Pathway, while Nucleotide Super-Pathway was predominant in semen samples with low quality (p Limitations, reasons for caution This is the first study presenting the entire metabolome signature of unprocessed human semen, and these preliminary results need to be confirmed in a bigger sample size. Wider implications of the findings Semen analyses applied in clinics do not evaluate the functional status of sperm, leaving the infertility causes due to male factor frequently unknown. Our study results could help to understand the molecular background of reduced seminal quality and male infertility and lead to identification of molecular biomarkers of functional sperm. Trial registration number not applicable

Context Despite the gut microbiome being widely studied in metabolic diseases, its role in polycystic ovary syndrome (PCOS) has been scarcely investigated. Objective Compare the gut microbiome in late fertile age women with and without PCOS and investigate whether changes in the gut microbiome correlate with PCOS-related metabolic parameters. Design Prospective, case–control study using the Northern Finland Birth Cohort 1966. Setting General community. Participants A total of 102 PCOS women and 201 age- and body mass index (BMI)-matched non-PCOS control women. Clinical and biochemical characteristics of the participants were assessed at ages 31 and 46 and analyzed in the context of gut microbiome data at the age of 46. Intervention (s): None Main outcome measure(s) Bacterial diversity, relative abundance, and correlations with PCOS-related metabolic measures. Results Bacterial diversity indices did not differ significantly between PCOS and controls (Shannon diversity P = .979, unweighted UniFrac P = .175). Four genera whose balance helps to differentiate between PCOS and non-PCOS were identified. In the whole cohort, the abundance of 2 genera from Clostridiales, Ruminococcaceae UCG-002, and Clostridiales Family XIII AD3011 group, were correlated with several PCOS-related markers. Prediabetic PCOS women had significantly lower alpha diversity (Shannon diversity P = .018) and markedly increased abundance of genus Dorea (false discovery rate = 0.03) compared with women with normal glucose tolerance. Conclusion PCOS and non-PCOS women at late fertile age with similar BMI do not significantly differ in their gut microbial profiles. However, there are significant microbial changes in PCOS individuals depending on their metabolic health.

STUDY QUESTION Does endometrium harbour functionally active microorganisms and whether the microbial composition differs between proliferative and mid-secretory phases? SUMMARY ANSWER Endometrium harbours functionally alive microorganisms including bacteria, viruses, archaea and fungi whose composition and metabolic functions change along the menstrual cycle. WHAT IS KNOWN ALREADY Resident microbes in the endometrium have been detected, where microbial dysfunction has been associated with reproductive health and disease. 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE With the novel metatranscriptomic approach, we mapped the entire alive microbiota composing of >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. LARGE SCALE DATA The raw RNAseq data used in the current study are available at GEO GSE86491 and at BioProject PRJNA379542. LIMITATIONS, REASONS FOR CAUTION These pioneering results should be confirmed in a bigger sample size. WIDER IMPLICATIONS OF THE FINDINGS 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., 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 Andalucía-Consejería de Economía y Conocimiento: MENDO (B-CTS-500-UGR18) and by the University of Granada Plan Propio de Investigacio ́n 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.

A recent paper in BMC Biology entitled “A tissue level atlas of the healthy human virome” by Kumata et al. describes a meta-transcriptomic analysis of RNA-sequencing datasets from the Genotype-Tissue Expression (GTEx) Project. Using a workflow that maps the GTEx sequences to the human genome, then screens unmapped sequences to detect viral transcripts, the authors present a quantitative analysis of the presence of different viruses in the nondiseased tissues of over 500 individuals and assess the impact of these viruses on host gene expression. Here we draw attention to an issue not acknowledged in this study. Namely, by relying solely on GTEx datasets, which are enriched for transcripts with poly(A) tails, the analysis will have missed non-poly(A) viral transcripts, rendering this tissue level atlas of the virome incomplete, Spanish Ministry of Economy, Industry and Competitiveness (MINECO), European Union (EU) RYC-2016-21199 ENDORE SAF2017-87526-R, FEDER/Junta de Andalucía -Consejería de Economía y Conocimiento: MENDO B-CTS-500-UGR18, University of Granada Plan Propio de Investigacion 2016 -Excellence actions: Unit of Excellence on Exercise and Health (UCEES) SOMM17/6107/UGR, Spanish Ministry of Science, Innovation and Universities PRE2018-0854409 FPU19/01638

There is growing evidence that the upper female genital tract is not sterile, harbouring its own microbial communities. However, the significance and the potential effect of endometrial microorganisms on reproductive functions remain to be fully elucidated. Analysing the endometrial microbiome, the microbes and their genetic material present in the endometrium, is an emerging area of study. The initial studies suggest it is associated with poor reproductive outcomes and with different gynaecological pathologies. Nevertheless, studying a low-biomass microbial niche as is endometrium, the challenge is to conduct well-designed and well-controlled experiments in order to avoid and adjust for the risk of contamination, especially from the lower genital tract. Herein, we aim to highlight methodological considerations and propose good practice recommendations for future endometrial microbiome studies.

We acknowledge the research support by Copan Italia S.p.A Inc., and Clearblue, SPD Swiss Precision Diagnostics GmbH. This study is part of a PhD Thesis conducted at the Official Doctoral Programme in Biomedicine of the University of Granada, Spain. We are grateful to Ana Yara Postigo-Fuentes for her assistance with English language., Current knowledge suggests that the uterus harbours its own microbiota, where the microbes could influence the uterine functions in health and disease; however, the core uterine microbial composition and the host-microbial relationships remain to be fully elucidated. Different studies are indicating, based on next-generation sequencing techniques, that microbial dysbiosis could be associated with several gynaecological disorders, such as endometriosis, chronic endometritis, dysfunctional menstrual bleeding, endometrial cancer, and infertility. Treatments using antibiotics and probiotics and/or prebiotics for endometrial microbial dysbiosis are being applied. Nevertheless there is no unified protocol for assessing the endometrial dysbiosis and no optimal treatment protocol for the established dysbiosis. With this review we outline the microbes (mostly bacteria) identified in the endometrial microbiome studies, the current treatments offered for bacterial dysbiosis in the clinical setting, and the future possibilities such as pro- and prebiotics and microbial transplants for modifying uterine microbial composition., 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; Programa Operativo FEDER Andalucía (B-CTS-500-UGR18) and by the University of Granada Plan Propio de Investigación 2016—Excellence actions: Unit of Excellence on Exercise and Health (UCEES)—and Plan Propio de Investigación 2018—Programa Contratos-Puente, and the Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades, European Regional Development Funds (ref. SOMM17/6107/UGR). A.S.-L. is funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-0854409).