The study of microorganisms is a field of great interest due to their environmental (e.g., soil contamination) and biomedical (e.g., parasitic diseases, autism) importance. The advent of ...revolutionary next-generation sequencing techniques, and their application to the hypervariable regions of the 16S, 18S or 23S ribosomal subunits, have allowed the research of a large variety of organisms more in-depth, including bacteria, archaea, eukaryotes and fungi. Additionally, together with the development of analysis software, the creation of specific databases (e.g., SILVA or RDP) has boosted the enormous growth of these studies. As the cost of sequencing per sample has continuously decreased, new protocols have also emerged, such as shotgun sequencing, which allows the profiling of all taxonomic domains in a sample. The sequencing of hypervariable regions and shotgun sequencing are technologies that enable the taxonomic classification of microorganisms from the DNA present in microbial communities. However, they are not capable of measuring what is actively expressed. Conversely, we advocate that metatranscriptomics is a "new" technology that makes the identification of the mRNAs of a microbial community possible, quantifying gene expression levels and active biological pathways. Furthermore, it can be also used to characterise symbiotic interactions between the host and its microbiome. In this manuscript, we examine the three technologies above, and discuss the implementation of different software and databases, which greatly impact the obtaining of reliable results. Finally, we have developed two easy-to-use pipelines leveraging Nextflow technology. These aim to provide everything required for an average user to perform a metagenomic analysis of marker genes with QIMME2 and a metatranscriptomic study using Kraken2/Bracken.
ABSTRACT The beneficial effects of physical activity (PA) on gut microbiome have been reported, nevertheless the findings are inconsistent, with the main limitation of subjective methods for ...assessing PA. It is well accepted that using an objective assessment of PA reduces the measurement error and also allows objective assessment of sedentary behavior (SB). We aimed to study the associations between accelerometer‐assessed behaviors (i.e., SB, light‐intensity physical activity LPA and moderate‐to‐vigorous physical activity MVPA) with the gut microbiome using compositional data analysis, a novel approach that enables to study these behaviors accounting for their inter‐dependency. This cross‐sectional study included 289 women from the Northern Finland Birth Cohort 1966. Physical activity was measured during 14 days by wrist‐worn accelerometers. Analyses based on the combined effect of MVPA and SB, and compositional data analyses in association with the gut microbiome data were performed. The microbial alpha‐ and beta‐diversity were not significantly different between the MVPA‐SB groups, and no differentially abundant microorganisms were detected. Compositional data analysis did not show any significant associations between any movement behavior (relative to the others) on microbial alpha‐diversity. Butyrate‐producing bacteria such as Agathobacter and Lachnospiraceae CAG56 were significantly more abundant when reallocating time from LPA or SB to MVPA ( γ = 0.609 and 0.113, both p ‐values = 0.007). While PA and SB were not associated with microbial diversity, we found associations of these behaviors with specific gut bacteria, suggesting that PA of at least moderate intensity (i.e., MVPA) could increase the abundance of short‐chain fatty acid–producing microbes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The female reproductive tract harbours unique microbial communities (known as microbiota) which have been associated with reproductive functions in health and disease. While endometrial microbiome ...studies have shown that the uterus possesses higher bacterial diversity and richness compared to the vagina, the knowledge regarding the composition of the Fallopian tubes (FT) is lacking, especially in fertile women without any underlying conditions.
To address this gap, our study included 19 patients who underwent abdominal hysterectomy for benign uterine pathology, and 5 women who underwent tubal ligation as a permanent contraceptive method at Hospital Clínico Universitario Virgen de la Arrixaca (HCUVA). We analyzed the microbiome of samples collected from the FT and endometrium using 16S rRNA gene sequencing.
Our findings revealed distinct microbiome profiles in the endometrial and FT samples, indicating that the upper reproductive tract harbors an endogenous microbiome. However, these two sites also shared some similarities, with 69% of the detected taxa Being common to both. Interestingly, we identified seventeen bacterial taxa exclusively present in the FT samples, including the genera
, and
, among others. On the other hand, 10 bacterial taxa were only found in the endometrium, including the genera
and
(FDR <0.05). Furthermore, our study highlighted the influence of the endometrial collection method on the findings. Samples obtained transcervically showed a dominance of the genus Lactobacillus, which may indicate potential vaginal contamination. In contrast, uterine samples obtained through hysterescopy revealed higher abundance of the genera
, and
.
Although the upper reproductive tract appears to have a low microbial biomass, our results suggest that the endometrial and FT microbiome is unique to each individual. In fact, samples obtained from the same individual showed more microbial similarity between the endometrium and FT compared to samples from different women. Understanding the composition of the female upper reproductive microbiome provides valuable insights into the natural microenvironment where processes such as oocyte fertilization, embryo development and implantation occur. This knowledge can improve
fertilization and embryo culture conditions for the treatment of infertility.
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.
