Endometrial Microbiota Research Articles

Interaction Between Functionally Activate Endometrial Microbiota and Host Gene Regulation in Endometrial Cancer.

Objective: In this study, we mainly explored two questions: Which microorganisms were functionally active in the endometrium of patients with endometrial cancer (EC)? What kind of response did the human host respond to functionally active microorganisms?Methods: Nine endometrial cancer patients and eight normal subjects were included in this study. HMP Unified Metabolic Analysis Network 3 (HUMAnN3) was used to obtain functional information of microorganisms. In addition, metaCyc-based GSEA functional enrichment analysis was used to obtain information on the metabolic pathways of the human host. At the same time, the O2PLS model and Spearman correlation analysis were used to analyze the microorganisms–host interaction.Results: With the novel metatranscriptome analysis pipeline, we described the composition of more than 5,000 functionally active microorganisms and analyzed the difference in microorganisms between the EC and the normal group. Our research found that these microorganisms were involved in part of the metabolic process of endometrial cancer, such as 6-sulfo-sialyl Lewis x epitope, N-acetyl-beta-glucosaminyl. In addition, the host–microbiota crosstalk of EC endometrium also included many biological processes, mainly functions related to tumor migration and the Apelin signaling pathway.Conclusion: The functionally active microorganisms in the EC endometrium played an essential role in the occurrence and migration of tumors. This meant that functionally active microorganisms could not be ignored in the treatment of endometrial cancer. This study helped to better understand the possible role of endometrial functional, active microorganisms in the occurrence and development of EC in patients with endometrial cancer and provided new information for new attempts to treat EC.

Integrated Analysis of Microbiome and Transcriptome Data Reveals the Interplay Between Commensal Bacteria and Fibrin Degradation in Endometrial Cancer.

The gut-uterus axis plays a pivotal role in the pathogenesis of endometrial cancer (EC). However, the correlations between the endometrial microbiome and endometrial tumor transcriptome in patients with EC and the impact of the endometrial microbiota on hematological indicators have not been thoroughly clarified. In this prospective study, endometrial tissue samples collected from EC patients (n = 30) and healthy volunteers (n = 10) were subjected to 16S rRNA sequencing of the microbiome. The 30 paired tumor and adjacent nontumor endometrial tissues from the EC group were subjected to RNAseq. We found that Pelomonas and Prevotella were enriched in the EC group with a high tumor burden. By integrating the microbiome and hematological indicators, a correlation was observed between Prevotella and elevated serum D-dimer (DD) and fibrin degradation products (FDPs). Further transcriptome analysis identified 8 robust associations between Prevotella and fibrin degradation-related genes expressed within ECs. Finally, the microbial marker of Prevotella along with DD and FDPs showed a high potential to predict the onset of EC (AUC = 0.86). Our results suggest that the increasing abundance of Prevotella in endometrial tissue combined with high serum DD and FDP contents may be important factors associated with tumor burden. The microbe-associated transcripts of host tumors can partly explain how Prevotella promotes DD and FDPs.

A PROTON PUMP INHIBITOR ALTERS THE pH OF THE HUMAN UTERINE CAVITY

To present two cases of increased pH of the uterine cavity after taking lansoprazole, an oral proton pump inhibitor (PPI). Case study. We examined infertile women to see whether chronic endometritis (CE) existed, through assessment of endometrial plasma cells (CD138 immunostained cells) and uterine microbiota by sequencing the 16S ribosomal RNA gene (Varinos Inc., Japan). CE was diagnosed applying the criteria (>5.15/10xmm2) reported by Liu et al. (1). We also examined the intrauterine pH directly on the endometrial surface using a Portal pH Meter (Japan Asch Inc., Japan) that has been utilized for the esophageal mucosa of esophagitis (2). We have reported in ESHRE annual meeting 2020 that intrauterine pH is weakly acidic (5.58±0.59) and it increases in the case of CE (6.67±0.39). Patient 1: A 44-year-old infertile woman noticed changes of her vaginal discharge after taking orally lansoprazole 30 mg for reflux esophagitis. Her initial intrauterine condition had been normal with pH: 6.3, Lactobacillus: 97%, and CD138-positive cells: 1.7/10xmm2. After PPI intake for a month, her intrauterine pH rose to 7.0, which suggested existence of CE. Therefore, we collected the endometrial tissue again and performed microbiota and pathological tests: Lactobacillus was 99% and CD138-positive cells were 1.6/10xmm2, which did not indicate CE histologically and bacterially. After discontinuing PPI, her intrauterine pH dropped to 6.7 with Lactobacillus: 97% and CD138-positive cells: 2.0/10xmm2. Patient 2: A 47-year-old woman, whose initial uterine pH had been 6.4, noticed a change of odor of vaginal discharge after taking PPI (lansoprazole 30 mg daily) for gastric ulcer. Her intrauterine pH rose to 7.1 with Lactobacillus :9.1% and CD138-positive cells: 0.7/10xmm2. After discontinuation of PPI, her intrauterine pH dropped to 6.2 with no CD138-positive cells, but Lactobacillus was 2.60%. We experienced two cases in which the pH of uterine cavity increased while taking oral PPI. We have reported that the uterine luminal pH in human is weakly acidic (around 6.0) and that the pH increases in the presence of CE or abnormal endometrial microbiota. In animals, such as mice (3) and mares (4), the uterine luminal pH is acidic at the time of implantation, and the acidic environment in the uterus is considered to be important for embryo implantation, and the vacuolar H + -ATPase pump, which is a type of proton pump, is expressed in the endometrium at the time of implantation in bovine (5) and mice (2), but there have been no reports in humans. These 2 cases which we presented here suggest that some proton pumps are also expressed in the endometrium and acidify the uterine cavity in humans, which could control the intrauterine environment independently of inflammation or microbiota.

O-127 The endometrial tissue microbiota of women who did or did not achieve a live birth within 12 months after a first failed IVF/ICSI cycle

Abstract Study question After one failed IVF/ICSI cycle, does the endometrial microbiota composition differ between women who will or will not reach a live birth within 12 months? Summary answer The endometrial microbiota composition did not significantly differ in women with one failed IVF/ICSI cycle with or without live birth, but statistical power was low. What is known already Evidence for the presence of an indigenous endometrial microbiome is mounting, and its composition may be associated with implantation success. However, a ‘core’ endometrial microbiome has not yet been defined, and its role in embryo implantation is still poorly understood. Further investigation of this topic may allow improvement and personalisation of clinical care for infertile couples. Endometrial microbiome analysis in infertile women has not yet been performed using transcervically obtained endometrial tissue. Using endometrial tissue instead of swabs or fluid may increase the bacterial DNA yield and therefore the precision of microbiome analyses. Study design, size, duration Endometrial tissue was obtained from a cohort of 141 infertile women undergoing endometrial scratching within a randomised controlled trial (RCT) (SCRaTCH trial, NL5193/NTR5342). Briefly, women aged 18-44 years with failed implantation after one full IVF/ICSI cycle and planning a subsequent IVF/ICSI cycle, were eligible. Participants were followed-up until 12 months after randomisation, with the primary outcome being live birth, defined as the delivery of at least one live foetus after 24 weeks of gestation. Participants/materials, setting, methods Endometrial tissue was obtained with an endometrial biopsy catheter in the midluteal phase of a natural cycle preceding subsequent IVF/ICSI, snap-frozen and stored at -80 °C until use. Total DNA was isolated from these biopsies, followed by 16S rRNA sequencing (V3-V4 region) to determine the endometrial microbiota composition. Positive (mock communities) and negative controls (DNA extraction and PCRs) were included. QIIME2 and DADA2 were used for the data analysis, followed by statistical analysis in R studio. Main results and the role of chance During the 12-month follow-up, 61/141 women (43.3%) reached a live birth. While endometrial microbiota profiles of all 141 women were analysed, only samples with ≥100 reads were included in the analysis, resulting in a total of 46 samples (32.6%) that were included in the analysis, which consisted of samples from 25 women who did not have and 21 women who did have a live birth within 12 months. The median number of reads per sample was not significantly different between the two groups (respectively 2,317 (IQR 651-19,031) and 1,335 (IQR 296-3,180), p = 0.29 by Mann-Whitney test). The endometrial microbiota detected, were bacterial genera frequently reported within the vaginal microbiota (e.g. Lactobacillus, Atopobium and Gardnerella). A clear dominance of Lactobacillus (relative abundance 55-100%, n = 22) or an unclassified bacterium genus (relative abundance 52-76%, n = 18) was observed in the majority of the samples; however, this dominance was not associated with the outcome of live birth. In addition, the samples dominated by Lactobacillus genera were mostly dominated by one species of Lactobacillus each (L. crispatus, L. iners, L. gasseri or L. jensenii). Limitations, reasons for caution The low biomass and the low ratio of bacterial versus human DNA in endometrial tissue were limiting factors in endometrial microbiota analysis. Furthermore, tissue was obtained transcervically, and contamination with vaginal/cervical microbiota could therefore have occurred. In the SCRaTCH trial no vaginal swabs were taken to serve as internal controls. Wider implications of the findings Future endometrial microbiota studies should consider the use of samples with a lower proportion of human DNA to maximize bacterial DNA yield. Furthermore, for endometrial microbiota research, sampling devices avoiding cervicovaginal contamination are desirable and may be developed in the future. Trial registration number SCRaTCH trial, NL5193/NTR5342

O-126 Endometrial microbiota composition is associated with reproductive outcome in infertile patients

Abstract Study question Is there an association between the composition of the endometrial microbiota and the reproductive outcomes in infertile patients undergoing in vitro fertilization (IVF)? Summary answer The composition of the endometrial microbiota (EM) prior to embryo transfer is associated with the different reproductive outcomes: live birth, no pregnancy or clinical miscarriage. What is known already The investigation of bacterial communities in the female reproductive tract using molecular methods has revealed the existence of a continuum microbiota that extends from the vagina to the upper genital tract. Previous evidence suggests the existence of an association between the vaginal and endometrial microbiome composition with reproductive and obstetrical outcomes. Specifically, the presence of specific pathogens together with low abundance of Lactobacilli has been associated with poor IVF outcomes. Study design, size, duration Multicentre prospective observational clinical study analysing the EM of infertile patients undergoing IVF (with maternal age ≤40) or ovum donation (≤50 years). A total of 452 infertile patients undergoing IVF/ovum donation were assessed for eligibility in 13 reproductive clinics in Europe, America, and Asia. The duration of the study was 30 months and the recruitment period extended between August 2017 and February 2019 (ct.gov 03330444). Participants/materials, setting, methods Endometrial fluid and endometrial biopsy were collected during a hormonal replacement therapy cycle after 5 days of progesterone (P) administration prior to a frozen embryo transfer cycle. Endometrial microbiota (EM) composition was analyzed using 16S rRNA gene sequencing using compositional data to transform scale-invariant values in both sample types. The EM in fluid and biopsy was associated with live birth, biochemical pregnancy, clinical miscarriage, or no pregnancy. Main results and the role of chance Of the 452 patients assessed, 44 did not meet the selection criteria and were excluded for the study and 66 patients were lost to follow-up. Of the 342 remaining patients, 198 (57.9%) became pregnant [141 (41.2%) had a live birth, 27 (7.9%) had a biochemical pregnancy, 2 (0.6%) had an ectopic pregnancy, and 28 (8.2%) a clinical miscarriage], while 144 (42.1%) did not become pregnant. The baseline characteristics, clinical and embryological variables were homogeneous and no bias toward the clinical outcome categories was observed. Our association study showed that the composition of the EM was associated with the reproductive outcome in both endometrial fluid and biopsy. A dysbiotic endometrial microbiota profile composed of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus was significantly associated with unsuccessful outcomes, especially no pregnancy and clinical miscarriage. In contrast, Lactobacillus was consistently enriched in patients with live birth outcomes. The EM in endometrial fluid did not fully reflect that in endometrial biopsy, although their association with clinical outcome was consistent. Limitations, reasons for caution The main limitation was the small number of biochemical pregnancy and clinical miscarriage analysed. During transcervical collection of endometrial samples caution was taken to avoid contamination with the cervix although cervical contamination cannot be fully discarded. Wider implications of the findings Our data indicate that EM dysbiosis is associated with poor clinical outcome in ART. Thus, the EM composition before embryo transfer could be a useful biomarker to consider offering an opportunity to further improve diagnosis and treatment strategies. Trial registration number Clinical trials.gov 03330444

P–333 Vaginal and endometrial microbiota: is there any correlation

Abstract Study question Is there any correlation between the total bacterial load and the lactobacilli quantities in the vaginal and endometrial microbiomes in reproductive-age women? Summary answer There was no correlation between the vaginal and endometrial total bacterial loads and only a weak positive correlation between the quantities of lactobacilli. What is known already The Lactobacilli-dominated microbiota is considered to be the most favorable type of microbiota in the uterine cavity. It is associated with increased reproductive success in women undergoing in vitro fertilization. Whereas the non-Lactobacillus dominated microbial communities are more frequent in women with poor pregnancy outcomes. When analyzing endometrial microbiota, one of the challenges is sampling. Transvaginal sample intake involves the possibility of contaminating the samples with vaginal microbiota. Moreover, it is an invasive procedure leading to the development of infectious inflammatory diseases of the upper genital tract. Thus, researchers are currently searching for predictors of the state of endometrial microbiota. Study design, size, duration It is a cross-sectional study of the vaginal end endometrial micorbiomes from 64 reproductive-age women. Endometrial and vaginal samples were collected simultaneously on days 7–10 of the menstrual cycle. To avoid contamination by vaginal microbiota, Endobrush Standard for Endometrial Cytology (Laboratoire C.C.D.; France) was used for endometrial sampling. Participants/materials, setting, methods The study included women who came to the “Garmonia” Medical Center (Yekaterinburg, Russia) seeking infertility treatment. The average age of the patients was 32.2±5.0. DNA from vaginal and endometrial samples was extracted using PREP-NA-PLUS kit (DNA-Technology, Russia). Vaginal and endometrial microbiota was analyzed using Femoflor real-time PCR kit and DTprime 4M1 thermocycler (DNA-Technology, Russia). Main results and the role of chance Total bacterial load (TBL) in vaginal discharge was 3.8–7.9 lg (median — 7.1, interquartile range — 6.6–7.4). TBL in the endometrial samples was 0–5.1 lg (median — 3.9, interquartile range — 3.6–4.2). There was no correlation between TBL values in vaginal discharge and endometrial samples (Spearman’s rho — 0.247, p = 0.049). Lactobacilli quantities in vaginal discharge were 4.5–8.3 lg (median — 7.2, interquartile range — 6.4–7.6), in endometrial samples — 0–5.1 lg (median — 3.7, interquartile range — 3.1–4.2). There was a weak positive correlation between lactobacilli quantities in vaginal and endometrial samples (Spearman’s rho — 0.362, p = 0.003). The proportion of lactobacilli in vaginal discharge was 1–100% (median — 100%, interquartile range — 95–100%), in the endometrial samples — 0–100% (median — 96%, interquartile range — 25–100%). There was no correlation between lactobacilli proportions in vaginal and endometrial samples (Spearman’s rho — 0.225, p = 0.074). Furthermore, there was no correlation between lactobacilli quantity in the vagina and their proportion in the endometrial microbiota (Spearman’s rho — 0.294, p = 0.018). There was only a weak positive correlation between the quantities of lactobacilli in vaginal and endometrial samples. Vaginal TBL values and lactobacilli proportions did not correlate with lactobacilli quantities and proportions in the endometrial samples. Limitations, reasons for caution The study was conducted on a small sample. Moreover, it is notoriously difficult to interpret the analysis results for endometrial microbiota due to the high risk of contamination and its low microbial biomass. Wider implications of the findxings: Apparently, there is no obvious link between the vaginal and endometrial microbiomes. It is possible that, apart from vaginal microbiota, there are other predictors which could allow us to assume whether lactobacilli are present in the endometrial microbiota. Trial registration number Not applicable

O-143 Characterization of vaginal and endometrial microbiome in patients with chronic endometritis (CE)

Abstract Study question Could vaginal and endometrial microbiome by sequencing 16S rRNA be comparable to classic diagnostic methods or immunohistochemistry CD138 for diagnosis of chronic endometritis? Summary answer A characteristic endometrial and vaginal microbiome is present in patients with chronic endometritis. An abnormal vaginal microbiome correlates with the presence of chronic endometritis. What is known already Chronic endometritis is a disease characterized by persistent inflammation of the endometrial lining. Currently, histopathological evaluation by immunohistochemistry CD138 marker is most common diagnostic method for CE. Microbiome analysis based on subunit 16S rRNA sequencing is a fast tool that can enable the identification of pathogenic microorganisms associated with CE. The main bacteria at vaginal and endometrial level belong to genus Lactobacillus, producers of lactic acid that allows maintaining acidic pH of vagina and acts as barrier against pathogens. Investigations on the effect of an abnormal endometrial and vaginal microbiome could improve assisted reproductive technologies. Study design, size, duration This is a observational pilot study (60 patients and 120 samples). The study population consists of patients attending to our fertility clinic for frozen euploid embryo transfer (FET) from May 2017 to May 2019. Preimplantation Genetic Testing of aneuploidy (PGT-A) was performed at blastocyst stage using Veriseq (Illumina). The inclusion criteria to be meet by patients were: age between 18 and 50 years, own or donated oocytes and use of ICSI. Participants/materials, setting, methods Cohort study with sixty patients undergoing assisted reproductive treatment (TRA) with their own or donated gametes and PGT-A Vaginal and endometrial samples were taken in the cycle prior to embryo transfer. The vaginal and endometrial microbiome was analyzed by mass sequencing of the V3V4 region of 16S rRNA. Bioinformatics analysis was performed using QIIME2 and MicrobiomeAnalyst packages. Alpha, beta diversity and taxonomic characterization were compared with positive and negative CD138 groups for chronic endometritis (CE). Main results and the role of chance Different bacterial communities were detected when vaginal and endometrial samples were analyzed in patients with and without endometritis diagnosed with CD138 immunohistochemistry. In patients with endometritis, a higher alpha diversity index tendency was found in vaginal samples (p = 0.15 for the Shannon index) and significant differences in endometrial samples (p = 0.01 for the Shannon index). In the beta diversity analysis, no significant differences were observed between the groups established as per the diagnosis of endometritis. Vaginal and endometrial samples from women with endometritis showed a microbiome pattern not dominated by Lactobacillus spp. Relative abundance analysis identified the genera Ralstonia and Gardnerella in endometrial sample, and the genera Streptoccoccus and Ureaplasma in vaginal sample of patients diagnosed with CD138 for endometritis. Comparing endometrial and vaginal samples CD138 positive diagnosed for endometritis, alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Relative abundance identified the genera Lactobacillus (p = 3.76E-4), Ralstonia (p = 8.19E-4), Delftia (p = 0.004) and Anaerobacillus (p = 0.004) in these sample groups. Limitations, reasons for caution The main limitation of this study is the small sample size. Larger studies including a higher number of samples are needed to confirm the different microbiome pattern observed at the vaginal and endometrial levels in correlation with chronic endometritis. The microbiome pattern has not been analyzed after treatment of CE. Wider implications of the findings Our findings suggest the existence of a characteristic vaginal and endometrial microbiota in patients with chronic endometritis. Different genera and species were identified in patients with and without endometritis depending on whether the sample was endometrial or vaginal. An abnormal vaginal microbiome appears to be strongly correlated with chronic endometritis. Trial registration number Not Applicable

O-128 Intra-cycle alterations of the uterine microbiota in patients with recurrent miscarriage or recurrent implantation failure and healthy controls

Abstract Study question Uterine microbiota: are there differences within three major time points of the menstrual cycle in healthy controls, recurrent miscarriage (RM) and recurrent implantation failure (RIF) patients? Summary answer Compared to controls, RM and RIF patients showed an altered uterine microbiota throughout the menstrual cycle, with a lower dominance of lactobacilli. What is known already In contrast to the former notion of a sterile womb, bacterial colonization in the uterus and the placenta has been demonstrated. Studies showed that Lactobacillus-dominated endometrial microbiota correlate with reproductive success. Moreover, the presence of non-Lactobacillus-dominated microbiota, especially with detection of Gardnerella and Streptococcus in the endometrial fluid, seems to be associated with lower implantation-, ongoing pregnancy- and live birth-rates. However, intra-cycle variations in healthy women as well as possible alterations in patients with RM or RIF remain unknown. Study design, size, duration In total, n = 20 RM patients (≥ 3 consecutive miscarriages), n = 20 RIF patients (≥3 fresh or frozen embryo transfers with negative serum hCG, good quality embryos) and n = 10 healthy controls (no pregnancy) were included in this study. All patients had a 28 day menstrual cycle. During follicular, ovulatory and luteal-phase, after a thorough cleaning of the cervix, a flexible catheter was introduced into the uterine cavity and a uterine flushing with 1ml of NaCl was performed. Participants/materials, setting, methods Bacterial DNA was extracted using a QIAamp DNA kit (Qiagen) in combination with a PrecellysR24 homogenizer (Peqlab, Erlangen, Germany) according to the manufacturer’s instructions. The V3-V4 region of the bacterial 16S rRNA gene was amplified. Samples were pooled in equimolar ratios and progressed to pyrosequencing using an Illumina MiSeq se-quencer with MiSeq Kit V2 (250 bp paired-end). Analysis of 16S rRNA data, including alpha- and beta-diversity, were calculated using the phyloseq package in R. Main results and the role of chance For the Shannon index (species richness and evenness) a significant decrease during the ovulation period was shown in the control group, indicating a more uniform microbiota (p < 0.05). This loss of diversity was not shown in RIF and RM patients. Overall, we could observe a higher similarity in taxonomic distribution in RM compared to the RIF patients. Longitudinal dynamics included increases in Firmicutes (CTRL and RM only) and a concomitant loss of Proteobacteria. Notably, significant amounts of bacteroides were only detected in the RIF patients. Actinobacteria were more frequent in both, RM and RIF as compared to controls. Limitations, reasons for caution To minimize the impact of a potential contamination, we performed pre-experiments with paired samples both from the vaginal fornix and the endometrial cavum and could show a significant difference in overall microbiome configuration. However, the route of sample can still be prone to contamination. Wider implications of the findings For the first time, we were able to show cycle-dependent alterations in the endometrial microbiome. These findings underline the role of an altered endometrial microbiome as a cause for RM and RIF and can contribute to the future establishment of therapeutic strategies in cases of a dysbalanced microbiome. Trial registration number Drks00020803

The impact of the female genital tract microbiome in women health and reproduction: a review.

The aim of this review is to gather the available research focusing on female genital tract (FGT) microbiome. Research question focuses in decipher which is the role of FGT microbiota in eubiosis, assisted reproduction techniques (ARTs), and gynaecological disorders, and how microbiome could be utilised to improve reproduction outcomes and to treat fertility issues. PubMed was searched for articles in English from January 2004 to April 2021 for "genital tract microbiota and reproduction", "endometrial microbiome", "microbiome and reproduction" and "microbiota and infertility". Manual search of the references within the resulting articles was performed. Current knowledge confirms predominance of Lactobacillus species, both in vagina and endometrium, whereas higher variability of species is both found in fallopian tubes and ovaries. Microbial signature linked to different disorders such endometriosis, bacterial vaginosis, and gynaecological cancers are described. Broadly, low variability of species and Lactobacillus abundance within the FGT is associated with better reproductive and ART outcomes. Further research regarding FGT microbiome configuration needs to be done in order to establish a more precise link between microbiota and eubiosis or dysbiosis. Detection of bacterial species related with poor reproductive outcomes, infertility or gynaecological diseases could shape new tools for their diagnosis and treatment, as well as resources to assess the pregnancy prognosis based on endometrial microbiota. Data available suggest future research protocols should be standardised, and it needs to include the interplay among microbiome, virome and mycobiome, and the effect of antibiotics or probiotics on the microbiome shifts.

Characterization of the vaginal and endometrial microbiome in patients with chronic endometritis

ObjectiveTo compare the endometrial and vaginal microbiome of women with and without chronic endometritis. Study designA cohort study with 60 patients undergoing assisted reproductive treatment with their own or donated gametes was undertaken. Vaginal and endometrial samples were taken in the cycle prior to embryo transfer. The endometrial and vaginal microbiome was analysed by mass sequencing of the V3V4 region of 16S rRNA gene. Bioinformatics analysis was performed using QIIME2 and MicrobiomeAnalyst packages. Alpha diversity, beta diversity and taxonomic characterization were compared between samples that tested positive and negative for chronic endometritis on CD138 immunohistochemistry. ResultsDifferent bacterial communities were detected when vaginal and endometrial samples were analysed in patients with and without endometritis diagnosed using CD138 immunohistochemistry. In patients with endometritis, a higher alpha-diversity index was found in vaginal samples (p = 0.15 for the Shannon index) and significant differences were found in endometrial samples (p = 0.01 for the Shannon index). In the beta-diversity analysis, no significant differences were observed between the groups with and without endometritis. Vaginal and endometrial samples from women with endometritis showed a microbiome pattern that was not dominated by Lactobacillus spp. Relative abundance analysis identified Ralstonia and Gardnerella spp. in endometrial samples, and Streptoccoccus and Ureaplasma spp. in vaginal samples of patients diagnosed with chronic endometritis on CD138 immunohistochemistry. When comparing endometrial and vaginal samples diagnosed with endometritis on CD138 immunohistochemistry, both alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Lactobacillus spp. (p = 3.76E-4), Ralstonia spp. (p = 8.19E-4), Delftia spp. (p = 0.004) and Anaerobacillus spp. (p = 0.004) were identified in these sample groups. ConclusionThese results demonstrate the existence of a characteristic vaginal and endometrial microbiota in patients with chronic endometritis. Different genera and species were identified in patients with and without chronic endometritis depending on whether the sample was endometrial or vaginal. There is a clear relationship between changes in the vaginal microbiome and chronic endometritis. The microbiota is a continuum throughout the female reproductive tract, so study of the vaginal microbiota could be useful for the diagnosis of diseases of the upper reproductive tract, such as chronic endometritis.

Ectopic pregnancies and endometrial microbiota.

In recent years there has been significant progress in the study of endometrial microbiota. This line of investigation has not been free of controversy and discussion. It is a key for clinicians involved to remain updated with the most recent findings in microbiome and its clinical implications to be able to offer patients all possible treatments. The existence of endometrial microbiota now seems undisputed. Current lines of work are centered on dysbiosis and its connection to other pathologies. It is in the field of assisted reproductive technology where this research plays an even more crucial role. In this case, we are focusing our attention toward the study of ectopic pregnancies, searching for similarities in their etiopathogenesis and alterations in the endometrial microbiota. Ectopic pregnancy has great repercussions for patients and for the healthcare system. We must continue researching to offer patients techniques and behaviors that can prevent it. The increase in its incidence makes ectopic pregnancy an entity that we must study.

Analysis of vaginal and endometrial microbiota communities in infertile women with a history of repeated implantation failure.

PurposeTo identify specific bacterial communities in vaginal and endometrial microbiotas as biomarkers of implantation failure by comprehensively analyzing their microbiotas using next‐generation sequencing.MethodsWe investigated α‐ and β‐diversities of vaginal and endometrial microbiotas using 16S rRNA gene sequencing and compared their profiles between 145 women with repeated implantation failure (RIF) and 21 controls who lacked the factors responsible for implantation failure with a high probability of being healthy and fertile to identify specific bacteria that induce implantation failure.ResultsThe endometrial microbiotas had higher α‐diversities than did the vaginal microbiotas (P < .001). The microbiota profiles showed that vaginal and endometrial samples in RIF patients had significantly higher levels of 5 and 14 bacterial genera, respectively, than those in controls. Vaginal Lactobacillus rates in RIF patients were significantly lower at 76.4 ± 38.9% compared with those of the controls at 91.8 ± 22.7% (P = .018), but endometrial Lactobacillus rates did not significantly differ between the RIF patients and controls (56.2 ± 36.4% and 58.8 ± 37.0%, respectively, P = .79).ConclusionsImpaired microbiota communities containing specific bacteria in both the endometrium and vagina were associated with implantation failure. The vaginal Lactobacillus rates, but not the endometrial, may be a biomarker for RIF.

UTERINE MICROBIOME IN HEALTHY FERTILE WOMEN AND IN CASE OF ENDOMETRIUM PATHOLOGY IN MULTIPLE FAILURES OF IN-VITRO-FERTILIZATION PROGRAMS

Background: Thanks to new diagnostic technologies, it is of no doubt that the uterus is no longer considered to be sterile. A disturbed microbiome of endometrium can be a significant reason for preterm birth, infertility, the recurrence of pregnancy loss, and repeated implantation failures in in-vitro-fertilization programs. Aim: Study the endometrial microbiome features in healthy fertile women and in women with multiple in-vitro-fertilization failures. Methods: To assess the differences in the endometrial microbiota of 20 women with infertility and experiencing multiple unsuccessful attempts of in-vitro-fertilization and 15 fertile, healthy women, endometrial microbiome was studied using an NGS of 16S rRNA. Results and Discussion: Lactobacillus (29.4%), Comamonas (16.8%), and Mesorhizobium (6.0%) were the most abundant genera in group of healthy fertile patients, and Lactobacillus (33.3%), Ralstonia (7.9%) and Pediococcus (4.8%) were most abundant in the group of infertile patients. The mean relative abundance of Lactobacillus did not significantly differ between groups and comprised 33.3% in the group of infertile women and 29.4% in healthy fertile women. A considerably higher mean relative abundance of bacteria of the genus Brevundimonas and Ralstonia was recorded in the group of infertile women. The fertile women had a statistically significantly higher mean relative abundance of Acidovorax, Brevibacillus, Caulobacter, Comamonas, Delftia, Distigma, Pseudomonas, Schlegelella, and Thermus. Conclusions: The presented data confirm that endometrium is not a sterile tissue despite long-standing concepts concerning its sterility. Lactobacillus are dominant genera; however, there is no absolute dominance of Lactobacillus over 90%. The mean relative abundance of Lactobacillus in the uterine microbiome in fertile patients and patients with multiple in-vitro-fertilization failures did not differ.

Mapping the entire functionally active endometrial microbiota.

Does endometrium harbour functionally active microorganisms and whether the microbial composition differs between proliferative and mid-secretory phases? Endometrium harbours functionally alive microorganisms including bacteria, viruses, archaea and fungi whose composition and metabolic functions change along the menstrual cycle. 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. 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. 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. 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. The raw RNAseq data used in the current study are available at GEO GSE86491 and at BioProject PRJNA379542. These pioneering results should be confirmed in a bigger sample size. 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 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.

Analysis of endometrial microbiota in intrauterine adhesion by high-throughput sequencing.

BackgroundIntrauterine adhesions (IUA) arise from scar tissue formation between the endometrial surfaces in response to mechanical or infectious injuries. However, the potential role of endometrial microbiota in IUA remains unclear. We aimed to explore the composition of endometrial microbiota and its potential role in IUA.MethodsWe retrospectively enrolled 46 patients diagnosed with IUA and 21 infertility patients without intrauterine lesions, as control subjects. All cases were diagnosed with hysteroscopy and endometrial tissues were taken from the intrauterine cavity using a hysteroscopic cutting ring without electricity study. After endometrial samples were collected, DNA was extracted and amplified for barcoded Illumina high-throughput next-generation sequencing targeted to the 16S rRNA V4 region for microbiota. Microbiota data were compared between two groups using α-diversity, β-diversity and Nonmetric Multidimensional Scaling based on Weighted Unifrac distance.ResultsAt the phyla level, the dominant bacteria included Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Proteobacteria accounted for more than 64.48%. At the genus level, the proportions of Klebsiella, Shewanella, and Lactobacillus were higher in patients with IUA than in non- IUA participants (20.67% and 8.77%, P=0.006, 13.37% and 4.53%, P=0.175, 12.74% and 6.95%, P=0.882; respectively). The proportion of Acinetobacter was significantly lower in patients with IUA than in non- IUA participants (P=0.005).ConclusionsEndometrial microbiota differ between patients with IUA and infertility patients without intrauterine lesions, and the potential variation of endometrial microbiota might cause IUA.

Хронический эндометрит. Пути улучшения методов диагностики

This paper reviews an important issue that significantly affects female fertility, chronic endometritis. This condition is characterized by clinical presentations of various severity and abnormalities of endometrial morphology. The rate of chronic endometritis varies greatly, mainly due to predominantly asymptomatic course and imperfect diagnostics. Pathogenic mechanisms of endometrial inflammation and different views on its effects on female reproductive potential are addressed. Further search for endometrial receptivity markers is needed as these markers will allow for assessing the severity of endometrial damage to develop the algorithm of preconception care. The authors focus on diagnostic techniques for chronic endometritis and their value. Immunohistochemistry of endometrium is a promising tool to be applied to diagnose endometrial inflammation and monitor treatment efficacy. A complex diagnostic approach to chronic endometritis that includes pelvic ultrasound, Doppler ultrasound, assessment of endometrial microbiota, diagnostic hysteroscopy and endometrial histopathology, and evaluation of endometrial receptivity is useful. KEYWORDS: chronic endometritis, termination of pregnancy, infertility, autoimmune processes, chronic inflammation, endometrial structure, immunohistochemistry. FOR CITATION: Shamilova A.M., Il’ina I.Yu., Borovkova E.I., Dobrokhotova Yu.E. Chronic endometritis. Towards the improvement of diagnostic methods. Russian Journal of Woman and Child Health. 2021;4(3):243–249 (in Russ.). DOI: 10.32364/2618-8430-2021-4-3-243-249.

Микробиом эндометрия у женщин с многократными неудачами экстракорпорального оплодотворения

Objective. To assess endometrial microbiome in women with infertility and multiple failed in vitro fertilization (IVF) cycles. Patients and methods. The study included 42 women; group 1 consisted of 22 women aged 20 to 42 with infertility and repeated unsuccessful IVF cycles; in group 2 (control), there were 20 healthy women aged 20 to 42 years, planning pregnancy. Microbiome samples for the study were taken from 20 to 24 days of the menstrual cycle. Results. Higher relative concentrations of Lactobacillus iners, Lactobacillus acidophilus, Lactobacillus jensenii, Lactobacillus crispatus, Prevotella melaninogenica, Bacteroides vulgatus, Corynebacterium bouchesdurhonense, Bacteroides caccae, Bifidobacterium gallinarum, Bifidobacterium adolescentis were revealed in the group of healthy women without aggravating factors in obstetric and gynecological medical history. Higher relative concentrations of Methylobacterium aerolatum and Comamonas testosteroni were found in women in group 1. A distinctive feature of endometrial microbiota in women in this group was the presence of Streptococcus spp. and Gardnerella vaginalis in low concentrations. The average relative representation of the genus Lactobacillus was 34.4% in group 1 and 63.0% in group 2. In general, the composition of the uterine microbiome contained bacteria characteristic of oral and intestinal biotopes. Conclusion. Women with multiple failed IVF cycles have greater biodiversity than healthy women. The presence of high concentrations of Lactobacillus may be a marker of favorable reproductive outcomes. Key words: endometrial microbiome, failure, in vitro fertilization, Lactobacillus

Dysbiosis of the endometrial microbiota and its association with inflammatory cytokines in endometrial cancer

The underlying molecular mechanisms involved in the pathogenesis of endometrial cancer (EC) are still not well understood. Our goal was to investigate the composition of the endometrial microbiota and the association with inflammatory cytokines in EC. Endometrial microbiota profiles of women with EC (n = 25) and benign uterine lesions (BUL, n = 25) were assessed by 16S ribosomal RNA gene amplicon sequencing. The expression levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-17 (IL-17) mRNA and protein in the endometrial tissues of the two groups were determined by real-time quantitative polymerase chain reaction and Western blot, respectively. There were significant differences in alpha diversity based on the observed operational taxonomic units (P = .002), Pielou evenness (P = .001), and Shannon index (P < .001) between EC and BUL groups. Significant differences were also found in Bray-Curtis (P = .001) and unweighted UniFrac (P = .001) beta diversity measures between the two groups. At the genus level, Micrococcus was more abundant in the EC group. Pseudoramibacter_Eubacterium, Rhodobacter, Vogesella, Bilophila, Rheinheimera, and Megamonas were enriched in the BUL group. There were no differences in IL-8 and IL-17 protein levels between the two groups, except IL-6 protein levels. However, the mRNA expression levels of IL-6, IL-8, and IL-17 were significantly different. Moreover, the relative abundances of Micrococcus was positively correlated with IL-6, and IL-17 mRNA levels. In conclusion, our results suggested that dysbiosis of endometrial microbiota and the inflammatory cytokines were associated with Micrococcus in EC patients, which might be useful for exploration of the mechanism between the endometrial microbiota and inflammatory responses in future studies.

Recent Insights on the Maternal Microbiota: Impact on Pregnancy Outcomes.

Hormonal changes during and after pregnancy are linked with modifications in the maternal microbiota. We describe the importance of the maternal microbiota in pregnancy and examine whether changes in maternal microbiotic composition at different body sites (gut, vagina, endometrium) are associated with pregnancy complications. We analyze the likely interactions between microbiota and the immune system. During pregnancy, the gastrointestinal (gut) microbiota undergoes profound changes that lead to an increase in lactic acid–producing bacteria and a reduction in butyrate-producing bacteria. The meaning of such changes needs clarification. Additionally, several studies have indicated a possible involvement of the maternal gut microbiota in autoimmune and lifelong diseases. The human vagina has its own microbiota, and changes in vaginal microbiota are related to several pregnancy-related complications. Recent studies show reduced lactobacilli, increased bacterial diversity, and low vaginal levels of beta-defensin 2 in women with preterm births. In contrast, early and healthy pregnancies are characterized by low diversity and low numbers of bacterial communities dominated by Lactobacillus. These observations suggest that early vaginal cultures that show an absence of Lactobacillus and polymicrobial vaginal colonization are risk factors for preterm birth. The endometrium is not a sterile site. Resident endometrial microbiota has only been defined recently. However, questions remain regarding the main components of the endometrial microbiota and their impact on the reproductive tract concerning both fertility and pregnancy outcomes. A classification based on endometrial bacterial patterns could help develop a microbiota-based diagnosis as well as personalized therapies for the prevention of obstetric complications and personalized treatments through nutritional, microbiotic, or pharmaceutical interventions.

Controlled ovarian stimulation and progesterone supplementation affect vaginal and endometrial microbiota in IVF cycles: a pilot study

PurposeDoes controlled ovarian stimulation (COS) and progesterone (P) luteal supplementation modify the vaginal and endometrial microbiota of women undergoing in vitro fertilization?MethodsFifteen women underwent microbiota analysis at two time points: during a mock transfer performed in the luteal phase of the cycle preceding COS, and at the time of fresh embryo transfer (ET). A vaginal swab and the distal extremity of the ET catheter tip were analyzed using next-generation 16SrRNA gene sequencing. Heterogeneity of the bacterial microbiota was assessed according to both the Bray-Curtis similarity index and the Shannon diversity index.ResultsLactobacillus was the most prevalent genus in the vaginal samples, although its relative proportion was reduced by COS plus P supplementation (71.5 ± 40.6% vs. 61.1 ± 44.2%). In the vagina, an increase in pathogenic species was observed, involving Prevotella (3.5 ± 8.9% vs. 12.0 ± 19.4%), and Escherichia coli-Shigella spp. (1.4 ± 5.6% vs. 2.0 ± 7.8%). In the endometrium, the proportion of Lactobacilli slightly decreased (27.4 ± 34.5% vs. 25.0 ± 29.9%); differently, both Prevotella and Atopobium increased (3.4 ± 9.5% vs. 4.7 ± 7.4% and 0.7 ± 1.5% vs. 5.8 ± 12.0%). In both sites, biodiversity was greater after COS (p < 0.05), particularly in the endometrial microbiota, as confirmed by Bray-Curtis analysis of the phylogenetic distance among bacteria genera. Bray-Curtis analysis confirmed significant differences also for the paired endometrium-vagina samples at each time point.ConclusionsOur findings suggest that COS and P supplementation significantly change the composition of vaginal and endometrial microbiota. The greater instability could affect both endometrial receptivity and placentation. If our findings are confirmed, they may provide a further reason to encourage the freeze-all strategy.