The female upper reproductive tract harbors endogenous microbial profiles.

Introduction: 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. Methods: 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. Results: 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 Enhydrobacter, Granulicatella, Haemophilus, Rhizobium, Alistipes, and Paracoccus, among others. On the other hand, 10 bacterial taxa were only found in the endometrium, including the genera Klebsiella, Olsenella, Oscillibacter and Veillonella (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 Acinetobacter, Arthrobacter, Coprococcus, Methylobacterium, Prevotella, Roseburia, Staphylococcus, and Streptococcus. Discussion: 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 in vitro fertilization and embryo culture conditions for the treatment of infertility.

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.

Comparison of Metagenomics and Metatranscriptomics Tools: A Guide to Making the Right Choice.

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.

Comparison of two identification and susceptibility test kits for Ureaplasma spp and Mycoplasma hominis in amniotic fluid of patients at high risk for intra-amniotic infection.

Objective: Ureaplasma urealyticum and Mycoplasma hominis are the most common microorganisms found in the amniotic fluid of patients at risk for preterm delivery. However, culture techniques for genital mycoplasms require special conditions, are barely considered as part of the evaluation of suspected intra-amniotic infection (IAI) and the results are available within 2 and 7 days. The objectives of this study are to validate the use of two commercially available kits (Mycoplasma IES y MYCOFAST® RevolutioN) for the identification of Ureaplasma spp. and Mycoplasma hominis in amniotic fluid, to compare the results of these kits with those obtained by culture and real-time polymerase chain reaction (qPCR) and to report the antibiotic sensitivity profile of the genital mycoplasms identified.Methods: This is a prospective cohort study including women with singleton and twin gestations between 16 and 36 weeks. Patients were admitted to perform an amniocentesis due to pregnancy complications considered at high risk for IAI (e.g. preterm labor with intact membranes, preterm prelabour rupture of membranes, short cervix, etc.), treatment of polyhydramnios, and for the assessment of fetal death and fever without a focus.Results: Overall, 93 patients underwent amniocentesis and 63 had results available for all tests. The prevalence of a positive culture was 6% (4/63). There were four cases of Ureaplasma spp. and none of Mycoplasma hominis. The qPCR identified one case as Ureaplasma spp., one case as Ureaplasma parvum and two cases as Ureaplasma urealyticum. For all tests, the diagnostic performance was as follows: sensitivity 100% [95% CI (39.8-100%)], specificity 100% [95% CI (93.9-100%)], positive predictive value 100% [95% CI (39.8-100%)] and negative predictive value 100% [95% CI (93.9-100%)]. In this cohort, Ureaplasma spp. showed low resistance to erythromycin, but a high resistance to clindamycin and clarithromycin that may change according to the antibiotic concentration.Conclusions: To our knowledge, this is the first study that validates the use of the Mycoplasma IES and MYCOFAST® RevolutioN kits for the identification of genital mycoplasmas in amniotic fluid. The results of these kits are mostly available within 24 hours, have an excellent correlation with those from broth cultures and qPCR and characterize the antibiotic sensitivity profile of the genital mycoplasms identified, providing an opportunity for specific treatment in cases of IAI. Further validation studies in other populations are needed.

Hallazgo de tripomastigoto en estudio citoquímico de líquido amniótico / Clinical finding of trypomastigote in citochemical study of amniotic fluid

Chagas disease is an important endemic disease in Latinamerica. The transmitions mechanism of Trypanosoma cruzi, etiologic agent of Chagas disease are multiples, this comunication is refering to congenital infection or transplacental infection. The aim of the present report was to describe the finding of trypomastigote in citochemical study of amniotic fluid from pregnant women with 32 weeks of gestation. Clinical findings of trypomastigote in amniotic fluid, shows the importance of microscopics tools in orger to detect and diagnose de Chagas disease in pregnant patients. Diagnosis of Chagas disease by citochemical study is an unreported and unexploited technique, easy to perform in an ordinary laboratory, allowing the possibility to explore events of early detection in the newborn