A systematic review identifying seminal plasma biomarkers and their predictive ability on IVF and ICSI outcomes.

The diverse nature and high molecule concentration of seminal plasma (SP) makes this fluid a good potential source for a potential biomarker that could predict assisted reproductive technology (ART) outcomes. Currently, semen quality parameters cannot accurately predict ART outcomes. A systematic literature search was conducted to identify human SP biomarkers with potential predictive ability for the outcomes of IVF and intracytoplasmic sperm injection. Observational cohort and case-control studies describing the association between biomarkers in human SP and the outcome of infertile men attending for ART were included. Forty-three studies were selected, reporting on 89 potential SP biomarkers (grouped as oxidative stress, proteins glycoproteins, metabolites, immune system components, metals and trace elements and nucleic acids). The present review supports 32 molecules in SP as potentially relevant biomarkers for predicting ART outcomes; 23 molecules were reported once and nine molecules were reported in more than one study; IL-18 and TGF-ß1-IL-18 ratio were confirmed in distinct studies. This review presents the most comprehensive overview of relevant SP biomarkers to predict ART outcomes to date, which is of clinical interest for infertility investigations and assisted reproduction; nevertheless, its potential is under-exploited. This review could serve as starting point for designing an all-encompassing study for biomarkers in SP and their predictive ability for ART outcomes, and for developing a non-invasive diagnostic tool.

Endometrial whole metabolome profile at the receptive phase: influence of Mediterranean Diet and infertility.

Introduction: Several metabolite classes have been identified in human endometrium, including lipids, nucleotides, amino acids, organic acids, and sugars. The first studies suggest the importance of metabolites in endometrial functions, as imbalance in uterine metabolites has been associated with low implantation rate and endometriosis. Nevertheless, most of studies have put emphasis on specific metabolite classes, and we lack the knowledge of the whole metabolome composition in human uterus. Further, a healthy dietary pattern has been shown to potentially protect against different endometrial dysfunctions and is a potential modulator of metabolomic composition and, consequently, the intrauterine microenvironment. The Mediterranean Diet (MD), characterized by a high intake of fruits, vegetables, cereals, nuts, legumes, fish, and olive oil, and a low consumption of meat, dairy products, and processed foods, has been associated with a wide range of benefits for health. Indeed, the MD pattern has displayed a beneficial role in endometriosis management and fertility; however, the relationship between the MD and the endometrial metabolome is still unknown. In our study, we set out to analyze receptive-phase endometrial metabolome profiles among women with infertility and their associations with MD. Methods: The study included women with male factor infertility (n=8), unexplained infertility (n=10), recurrent implantation failure (n=14), and endometriosis (n=13). The endometrial metabolome was analyzed with ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). The MD adherence of the participants was assessed using the 14-point MEDAS questionnaire of adherence to the MD. Results: We provide the whole metabolome profile of the endometrium, where 925 different metabolites were identified. Among these metabolites, lipids comprised the largest part, where polyunsaturated fatty acids (PUFAs) prevailed. Women with endometriosis and recurrent implantation failure were found to have lower levels of PUFAs compared to women with male factor and unexplained infertility (i.e., no clear endometrial alterations), identifying a metabolome profile associated with infertility diagnoses where altered endometrial functions are suspected. Moreover, MD adherence seemed to be associated with the endometrial metabolomic profile in a manner dependent on the health status of the uterus. Conclusion: The study findings provide insight into the molecular background of female infertility and lead to identification of potential molecular biomarkers and possibilities for modulating the endometrial microenvironment and, thereby, endometrial functions involved in embryo implantation and infertility.

Microbial composition across body sites in polycystic ovary syndrome: a systematic review and meta-analysis.

Polycystic ovary syndrome (PCOS) is an endocrine disorder affecting reproductive-aged women, but the cause remains unclear. Recent evidence has linked microbial composition with PCOS; however, the results are inconsistent. The aim of this systematic review was to gather current knowledge of the microbes across body sites (oral cavity, blood, vagina/cervix, gut) in women with PCOS, and meta-analyse the microbial diversity in PCOS. For this purpose, a systematic search using PubMed, Web of Science, Cochrane and Scopus was carried out. After selection, 34 studies met the inclusion criteria. Most of the studies associated changes in the microbiome with PCOS, whereas heterogeneity of the studies in terms of ethnicity, body mass index (BMI) and methodology, among other confounders, made it difficult to corroborate this relationship. In fact, 19 out of 34 of the studies were categorised as having high risk of bias when the quality assessment was conducted. Our meta-analysis on the gut microbiome of 14 studies demonstrated that women with PCOS possess significantly lower microbial alpha diversity compared with controls (SMD = -0.204; 95% CI -0.360 to -0.048; P = 0.010; I2 = 5.508, by Shannon Index), which may contribute to the development of PCOS. Nevertheless, future studies should specifically overcome the shortcomings of the current studies by through well planned and conducted studies with larger sample sizes, proper negative and positive controls and adequate case-control matching.

Exploring the Microbiome in Human Reproductive Tract: High-Throughput Methods for the Taxonomic Characterization of Microorganisms.

Microorganisms are important due to their widespread presence and multifaceted roles across various domains of life, ecology, and industries. In humans, they underlie the proper functioning of multiple systems crucial to well-being, including immunological and metabolic functions. Emerging research addressing the presence and roles of microorganisms within human reproduction is increasingly relevant. Studies implementing new methodologies (e.g., to investigate vaginal, uterine, and semen microenvironments) can now provide relevant insights into fertility, reproductive health, or pregnancy outcomes. In that sense, cutting-edge sequencing techniques, as well as others such as meta-metabolomics, culturomics, and meta-proteomics, are becoming more popular and accessible worldwide, allowing the characterization of microbiomes at unprecedented resolution. However, they frequently involve rather complex laboratory protocols and bioinformatics analyses, for which researchers may lack the required expertise. A suitable pipeline would successfully enable both taxonomic classification and functional profiling of the microbiome, providing easy-to-understand biological interpretations. However, the selection of an appropriate methodology would be crucial, as it directly impacts the reproducibility, accuracy, and quality of the results and observations. This review focuses on the different current microbiome-related techniques in the context of human reproduction, encompassing niches like vagina, endometrium, and seminal fluid. The most standard and reliable methods are 16S rRNA gene sequencing, metagenomics, and meta-transcriptomics, together with complementary approaches including meta-proteomics, meta-metabolomics, and culturomics. Finally, we also offer case examples and general recommendations about the most appropriate methods and workflows and discuss strengths and shortcomings for each technique.

Assessing the testicular sperm microbiome: a low-biomass site with abundant contamination.

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.

Analysing endometrial microbiome: methodological considerations and recommendations for good practice.

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.

Mapping the entire functionally active endometrial microbiota.

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. STUDY FUNDING/COMPETING INTERESTS: 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 Investigación 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.

The Gut Microbiome in Polycystic Ovary Syndrome and Its Association with Metabolic Traits.

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.

Omission of non-poly(A) viral transcripts from the tissue level atlas of the healthy human virome.

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 non-diseased 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.A commentary on Kumata et al. (BMC Biol 18:55, 2020).

New Opportunities for Endometrial Health by Modifying Uterine Microbial Composition: Present or Future?

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.