Endometrial microbiota composition is associated with reproductive outcome in infertile patients.

BACKGROUND: Previous evidence indicates associations between the female reproductive tract microbiome composition and reproductive outcome in infertile patients undergoing assisted reproduction. We aimed to determine whether the endometrial microbiota composition is associated with reproductive outcomes of live birth, biochemical pregnancy, clinical miscarriage or no pregnancy. METHODS: Here, we present a multicentre prospective observational study using 16S rRNA gene sequencing to analyse endometrial fluid and biopsy samples before embryo transfer in a cohort of 342 infertile patients asymptomatic for infection undergoing assisted reproductive treatments. RESULTS: A dysbiotic endometrial microbiota profile composed of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus was associated with unsuccessful outcomes. In contrast, Lactobacillus was consistently enriched in patients with live birth outcomes. CONCLUSIONS: Our findings indicate that endometrial microbiota composition before embryo transfer is a useful biomarker to predict reproductive outcome, offering an opportunity to further improve diagnosis and treatment strategies. Video Abstract.

The first glimpse of the endometrial microbiota in early pregnancy.

Investigation of the microbial community in the female reproductive tract with the use of sequencing techniques has revealed that endometrial samples obtained through a transvaginal catheter are dominated by Lactobacillus species. Dysbiotic changes in the endometrial microbiota may be associated with implantation failure or early spontaneous abortion in patients who undergo assisted reproductive technology treatment. Whether or not there is an endometrial microbiota in early pregnancy is unknown. Herein we describe, the human endometrial microbiota in a patient who subsequently had an 8th week spontaneous clinical miscarriage with euploid embryos in the next cycle and, for the first time, during a successful pregnancy in which the endometrial fluid was sampled at 4 weeks of gestation. The microbial profile found on the endometrial sample before the spontaneous abortion had higher bacterial diversity and lower Lactobacillus abundance than the endometrial fluid from the healthy pregnancy. Functional metagenomics detected different Lactobacillus species between the 2 samples. Lactobacillus crispatus was present in the endometrium before the spontaneous abortion, as were other bacteria involved in dysbiosis, which had an unstable functional pattern characterized by transposases and insertion elements. Lactobacillus iners was the most prevalent microbe found in the endometrium during early pregnancy; its presence was associated with defense mechanisms and basal functions. These novel observations prompt future investigations to understand the potential implications of microbiology on healthy and pathologic human pregnancy.

Taxonomical and Functional Assessment of the Endometrial Microbiota in A Context of Recurrent Reproductive Failure: A Case Report.

Investigation of the microbial community in the female reproductive tract has revealed that the replacement of a community dominated by Lactobacillus with pathogenic bacteria may be associated with implantation failure or early spontaneous abortion in patients undergoing assisted reproductive technology (ART) treatment. Herein we describe taxonomically and functionally the endometrial microbiome of an infertile patient with repeated reproductive failures (involving an ectopic pregnancy and two clinical miscarriages). The microbiological follow-up is presented over 18-month in which the microbiota was evaluated in six endometrial fluid samples. The microbial profile of 16S rRNA gene sequencing showed a persistent infection with Gardnerella and other bacterial taxa such as Atopobium and Bifidobacterium. In addition, taxonomic and functional analysis by whole metagenome sequencing in the endometrial fluid sample collected before one clinical miscarriage suggested the presence of multiple Gardnerella vaginalis clades with a greater abundance of clade 4, usually associated with metronidazole resistance. These results revealed a persistent G. vaginalis endometrial colonization presenting genetic features consistent with antimicrobial resistance, biofilm formation, and other virulence factors, which could be related to the reproductive failure observed.

Selection of New Probiotics for Endometrial Health.

Microbiota is a crucial player in gynecologic health, in which bacteria can shift to a dysbiotic state triggering a pathogenic process. Based on an ecological understanding of the problem, the aim of this study is to select a potential probiotic strain to improve female reproductive tract based on its capacity to initially lower pH and to promote the reduction of pathogenic bacteria. Based on this rationale, strain Lactobacillus rhamnosus BPL005 was initially selected for its capacity to reduce in vitro pH levels and produce organic acids. Subsequently, strain L. rhamnosus BPL005 (CECT 8800) was demonstrated to have a protective role on endometrial infections in an in vitro model of bacterial colonization of primary endometrial epithelial cells with Atopobium vaginae, Gardnerella vaginalis, Propionibacterium acnes, and Streptococcus agalactiae. In this model, BPL005 when co-cultured with those pathogens was shown to lower pH and to produce organic acids, being lactic acid the most relevant. The co-cultivation of strain L. rhamnosus BPL005 with tested reference pathogens produced a significant reduction in P. acnes and St. agalactiae levels and a non-significant reduction in A. vaginae and G. vaginalis. The colonization of L. rhamnosus BPL005 in the culture decreased IL-6, IL-8, and MCP-1, heightened in the presence of pathogens, and increased IL-1RA and IL-1 beta. Finally, safety was evaluated showing no signs of cytotoxicity, irritation in vaginal tests, or allergic contact dermatitis potential through the Local Lymph Node Assay. Overall, these results show the potential of L. rhamnosus BPL005 strain as a probiotic in gynecological health.

MicroRNA-30d deficiency during preconception affects endometrial receptivity by decreasing implantation rates and impairing fetal growth.

BACKGROUND: Maternal-embryonic crosstalk between the endometrium and the preimplantation embryo is required for normal pregnancy. Our previous results demonstrated that maternal microRNAs secreted into the endometrial fluid, specifically miR-30d, act as a transcriptomic regulator of the preimplantation embryo by the maternal intrauterine environment. OBJECTIVE: To investigate the reproductive and fetal effects of murine miR-30d deficiency at the maternal-embryonic interface according to the origin of its maternal or embryonic default. STUDY DESIGN: A miR-30d knockout murine model was used as the animal model to investigate the impact of maternal and/or embryonic origin of miR-30d deficiency on embryonic implantation and fetal development. Wild-type and miR-30d knockout pseudopregnant mice were used to study the effect of miR-30d deficiency on the receptivity markers by means of real-time quantitative polymerase chain reaction, immunofluorescence, and western blotting. We assessed receptivity markers and implantation rates in 6 different transfer conditions in which embryos obtained from wild-type, knockout, and knockout embryos pretreated with a miR-30d analog were transferred into either wild-type or knockout pseudopregnant females. The impact of miR-30d deficiency on fetal development was evaluated by analyzing the implantation sites and resorbing sites under physiological conditions at days 5, 6, 8, and 12 of pregnancy. Fetal growth was evaluated by analyzing fetuses and placentas at days 12 and 16 of pregnancy. RESULTS: Maternal miR-30d deficiency induced a significant downregulation of endometrial receptivity markers. In wild-type recipients, miR-30d knockout embryos had poorer implantation rates than wild-type embryos (48.86 ± 14.33% vs 75.00 ± 10.47%, respectively, P = .0061). In miR-30d knockout recipients, the lowest implantation rate was observed when knockout embryos were transferred compared to wild-type embryos (26.04 ± 7.15% and 49.71 ± 8.59%, respectively, P = .0059). A positive correlation (r = 0.9978) was observed for maternal leukemia inhibitor factor expression with implantation rates. Further, the course of gestation was compromised in miR-30d knockout mothers, which had smaller implantation sites, greater rates of resorption, and fetuses with smaller crown-rump length and fetal/placental weight ratio. CONCLUSION: Our results demonstrate that maternal and/or embryonic miR-30d deficiency impairs embryonic implantation and fetal development in the animal model. This finding adds a novel miRNA dimension to the understanding of pregnancy and fetal growth restriction in humans.

The diagnosis of chronic endometritis in infertile asymptomatic women: a comparative study of histology, microbial cultures, hysteroscopy, and molecular microbiology.

BACKGROUND: Chronic endometritis is a persistent inflammation of the endometrial mucosa caused by bacterial pathogens such as Enterobacteriaceae, Enterococcus, Streptococcus, Staphylococcus, Mycoplasma, and Ureaplasma. Although chronic endometritis can be asymptomatic, it is found in up to 40% of infertile patients and is responsible for repeated implantation failure and recurrent miscarriage. Diagnosis of chronic endometritis is based on hysteroscopy of the uterine cavity, endometrial biopsy with plasma cells being identified histologically, while specific treatment is determined based on microbial culture. However, not all microorganisms implicated are easily or readily culturable needing a turnaround time of up to 1 week. OBJECTIVE: We sought to develop a molecular diagnostic tool for chronic endometritis based on real-time polymerase chain reaction equivalent to using the 3 classic methods together, overcoming the bias of using any of them alone. STUDY DESIGN: Endometrial samples from patients assessed for chronic endometritis (n = 113) using at least 1 or several conventional diagnostic methods namely histology, hysteroscopy, and/or microbial culture, were blindly evaluated by real-time polymerase chain reaction for the presence of 9 chronic endometritis pathogens: Chlamydia trachomatis, Enterococcus, Escherichia coli, Gardnerella vaginalis, Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, Staphylococcus, and Streptococcus. The sensitivity and specificity of the molecular analysis vs the classic diagnostic techniques were compared in the 65 patients assessed by all 3 recognized classic methods. RESULTS: The molecular method showed concordant results with histological diagnosis in 30 samples (14 double positive and 16 double negative) with a matching accuracy of 46.15%. Concordance of molecular and hysteroscopic diagnosis was observed in 38 samples (37 double positive and 1 double negative), with an accuracy of 58.46%. When the molecular method was compared to microbial culture, concordance was present in 37 samples (22 double positive and 15 double negative), a matching rate of 56.92%. When cases of potential contamination and/or noncultivable bacteria were considered, the accuracy increased to 66.15%. Of these 65 patients, only 27 patients had consistent histological + hysteroscopic diagnosis, revealing 58.64% of nonconcordant results. Only 13 of 65 patients (20%) had consistent histology + hysteroscopy + microbial culture results. In these cases, the molecular microbiology matched in 10 cases showing a diagnostic accuracy of 76.92%. Interestingly, the molecular microbiology confirmed over half of the isolated pathogens and provided additional detection of nonculturable microorganisms. These results were confirmed by the microbiome assessed by next-generation sequencing. In the endometrial samples with concordant histology + hysteroscopy + microbial culture results, the molecular microbiology diagnosis demonstrates 75% sensitivity, 100% specificity, 100% positive and 25% negative predictive values, and 0% false-positive and 25% false-negative rates. CONCLUSION: The molecular microbiology method describe herein is a fast and inexpensive diagnostic tool that allows for the identification of culturable and nonculturable endometrial pathogens associated with chronic endometritis. The results obtained were similar to all 3 classic diagnostic methods together with a degree of concordance of 76.92% providing an opportunity to improve the clinical management of infertile patients with a risk of experiencing this ghost endometrial pathology.