Uterine Microbiome Research Articles

Impact of Lactobacillus in the uterine microbiota on in vitro fertilization outcomes

Abundant intrauterine Lactobacillus is associated with good in vitro fertilization (IVF) outcomes; however, whether specific species of Lactobacillus have any benefit remains unclear. So we examine the effect of Lactobacillus on the clinical outcomes of IVF at the species level. Uterine microbiota were classified as either Lactobacillus-dominant (LD) or non-Lactobacillus-dominant. In the LD group, we further investigated the clinical results for each Lactobacillus species and evaluated them in relation to IVF outcomes. In Uterine microbiome analysis, Lactobacillus was the most abundant, with the four species of L. crispatus, L. iners, L. gasseri, and L. jensenii accounting for the great majority. We compared the clinical outcomes of single frozen-thawed embryo transfer conducted by Lactobacillus species and found that the implantation rate was lowest in those in whom L. iners was dominant. This study is the first to conduct a species-level analysis of the uterine microbiota and report on a detailed investigation of Lactobacillus, which was believed to be particularly helpful for pregnancy.

An updated view on the role of the female reproductive tract microbiome in IVF outcomes

Introduction. In the last decade, a high-throughput 16S ribosomal RNA (rRNA) sequencing allowed to markedly extend insights into female reproductive tract microbiome. However, evidence about its role on in vitro fertilization (IVF) outcomes remains scarce and controversial.Aim: to analyze literature data for assessing an impact of the vaginal, uterine, and ovarian microbiome on IVF outcomes.Materials and Methods. The review was carried out based on publications from available in PubMed/MEDLINE, EBSCO, ResearchGate, Google Academy and еLibrary databases released over the last 20 years. For this, there was search for keywords and their combinations in Russian- and English-written publications: «vaginal microbiome», «cervical microbiome», «uterine microbiome», «ovarian microbiome», «microbiome of ovarian follicles», «IVF», «miscarriage», «early pregnancy loss», «implantation failure», «ovarian failure», «inflammasome». Only full-text original articles and reviews published in peer-reviewed journals were included in the review. Exclusion criteria were as follows: studies containing less than 10 observations per group, abstracts of conferences, studies on the male reproductive tract microbiome. Duplicate publicationswere excluded. The publications were selected independently by two co-authors, and in case of discrepancy two other co-authors were involved.Results. Vaginal, uterine, and ovarian microbiomes were characterized by providing relevant classifications and the features related to implantation failures and pregnancy loss after IVF. It was found that a decline in total Lactobacillus level and elevated proportion of L. jensenii, G. vaginalis, and Proteobacteria in the vaginal microbiome were predictors of implantation failure. In addition, IVF failure was also associated with the presence of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Streptococcus, Haemophilus, Staphylococcus, Brevundimonas, and Ralstonia in the uterine cavity as well as Bifidobacterium, Gardnerella, and Klebsiella in the endometrial tissue. On the other hand, Lactobacillus dominance in the uterine microbiome has a favorable effect. The colonization of the follicle by any microorganisms as well as the presence of follicular fluid anaerobic bacteria-derived metabolite trimethylamine-N-oxide (TMAO) associated with bacterial vaginosis additionally alters IVF outcomes. Moreover, the role of infectious cues in lowered ovarian reserve has also been established. Activation of the NLRP3 (NLR Family Pyrin Domain Containing 3) inflammasome by microbe-derived ligands stimulates production of pro-inflammatory cytokines and contributes to reduced follicle number. Blocking NLRP3 in mouse experiments can delay depletion of the follicle pool and result in elevated fertility.Conclusion. Favorable IVF outcomes are associated with Lactobacillus predominance in the vaginal and endometrial microbiome as well as lack of follicular fluid microorganisms. TMAO detected in the follicular fluid as well as activated NLRP3 inflammasome serve as negative predictors of IVF outcomes.

Influence of microbial colonization of the endometry on its function and development of hyperproliferative conditions

Abstract. The article provides an overview of literary sources that describe research on the microbiome of the endometrium in women of reproductive age. Thus, in many works, data is given indicating that the uterine cavity is not sterile. Various microorganisms can be found on the surface of the endometrium. So far, there is not enough research on microorganisms that can be considered a normal microbiome of the endometrium and its influence on the function and development of hyperproliferative processes of the endometrium in women. Many studies have proven the undoubted role of the uterine microbiome in the development of endometrial hyperplasia and other proliferative diseases. The cited studies indicate that 60 % of examined women with signs of microbial colonization caused by an infection of viral, bacterial, or fungal origin developed endometrial dysfunction, which led to the development of hyperproliferative processes. The study of the microbiocenosis of the uterine cavity in patients of reproductive age with various types of endometrial pathology indicates the role of certain pathogenic microflora in their occurrence. In all cases of atypical proliferation of the endometrium, an increase in the number of anaerobes up to 30 % was found among all isolated microorganisms, in particular, anaerobic bacteria of the genus Bacteroides spp. Representatives of the Enterobacteriaceae family (E. coli) and Gram-positive cocci (staphylococci and streptococci) prevailed among the aerobic flora. The largest spectrum of isolated microorganisms was found in patients with endometrial polyps. Studies of the immune system of the endometrium, which is formed to a greater extent due to the microbiome and directly participates in the cyclic changes of the endometrium, which are necessary for its physiological function during reproduction, are also presented. The immune system of the endometrium participates in the cyclic changes of the endometrium necessary for its physiological function in the process of reproduction. A proven factor is cells of the immune system and proper remodeling of spiral arteries, NK (neutrophil killer), T-lymphocytes and antigen-presenting cells (APC – antigen-presenting cell). A very important factor in the interaction between the gut microbiome and the immune system is the gut mucosa, and from this it can be inferred that similar connections may exist for the endometrium and its microbiome. Further studies of the state of the microbiome of the uterine cavity will allow adding information about its participation in functional processes and the pathogenesis of the development of hyperproliferative endometrial conditions.

Genital tract microbiome dynamics are associated with time of Chlamydia infection in mice

We have previously shown that the time of Chlamydia infection was crucial in determining the chlamydial infectivity and pathogenesis. This study aims to determine whether the time of Chlamydia infection affects the genital tract microbiome. This study analyzed mice vaginal, uterine, and ovary/oviduct microbiome with and without Chlamydia infection. The mice were infected with Chlamydia at either 10:00 am (ZT3) or 10:00 pm (ZT15). The results showed that mice infected at ZT3 had higher Chlamydia infectivity than those infected at ZT15. There was more variation in the compositional complexity of the vaginal microbiome (alpha diversity) of mice infected at ZT3 than those mice infected at ZT15 throughout the infection within each treatment group, with both Shannon and Simpson diversity index values decreased over time. The analysis of samples collected four weeks post-infection showed that there were significant taxonomical differences (beta diversity) between different parts of the genital tract—vagina, uterus, and ovary/oviduct—and this difference was associated with the time of infection. Firmicutes and Proteobacteria were the most abundant phyla within the microbiome in all three genital tract regions for all the samples collected during this experiment. Additionally, Firmicutes was the dominant phylum in the uterine microbiome of ZT3 Chlamydia infected mice. The results show that the time of infection is associated with the microbial dynamics in the genital tract. And this association is more robust in the upper genital tract than in the vagina. This result implies that more emphasis should be placed on understanding the changes in the microbial dynamics of the upper genital tract over the course of infection.

Metagenomic characterization of the equine endometrial microbiome during anestrus

Recent studies have confirmed that healthy uteri, including the equine uterus, harbour a microbiome. Studies on mares have been performed in estrus and therefore under the influence of high estrogen concentrations. Multiple human studies investigating the vaginal, as well as the gut microbiome have shown that the microbiome is dynamic throughout the estrous cycle and is influenced by differences in estrogen levels. This is in contrast to a study performed on Arabian mares which indicated that the equine vaginal microbiome is stable throughout the estrous cycle.In this study the resident uterine microbiome of healthy anestrous mares was characterized by metagenomics from double-guarded endometrial swabs and compared to the microbiome in the same mares during estrus.Double guarded endometrial swabs from 15 healthy estrous mares were taken and stored at -80°C until extraction, in the following non-breeding season 8 mares from this group were sampled in similar fashion during anestrus. Blank samples were obtained during both sample period. Genomic DNA was extracted using Qiagen DNeasy PowerSoil extraction kits and 16S rRNA gene was amplified using primers 515f-806r. An amplicon sequence variant table was made and chimeras were removed and the genomic16S rRNA from the blank samples was substracted. Sequences were processed and analysed using 16S rRNA SILVA v138.1 database. Contaminating reads were removed from the samples by subtracting the reads from the blank samples using Microdecon and downstream analysis performed using the Phyloseq package. Alpha and beta diversity were calculated using microbiome, amplicon and vegan packages.Results suggest that the equine uterine microbiome in estrus has a low diversity, low richness, and a low abundance at phylum level. The equine uterine microbiome in anestrus has a higher diversity, higher richness, and higher abundance compared to the estrus uterine microbiome. However, both the estrus as well as the anestrus endometrial microbiome is dominated by Proteobacteria, Firmicutes, and Bacteroidota. PCoA analysis displays a strong dissimilarity between estrus and anestrus samples.The difference in the microbiome between estrus and anestrus can possibly be explained due to the variance in immune responsiveness of the endometrium. The estrous mare's endometrium is under the influence of estrogens, allowing for a high immune response and possibly less diverse microbiome.

Association of the uterine microbiome to mare fertility

Currently, limited information on the microbiome (community of microorganisms) and its role in infertility in the equine uterus is available beyond the detection of specific pathogens in some subfertile mares. Given that less than 1% of environmental bacteria are cultivable on common media (Handelsman, J. 2004, Microbiol Mol Biol Rev 68, 669-685.), it is likely that these cultured species represent a minority of the resident population. The overarching objectives of this project were to i) identify the resident uterine microbiome in estrus ii) assess how this microbiome evolves over time and iii) compare traditional culture-dependent methods of assessing the presence of bacteria in the equine uterus with 16S rRNA sequencing. Uterine cytobrush and swab samples were collected from 157 maiden Thoroughbred mares aged 8 years or younger and 116 Thoroughbred barren mares aged 4 to 15 years at the first reproductive exam of the breeding season. A second sample was collected from mares that were not pregnant at the end of the season (n=23). Furthermore, 20 of the mares were sampled in three consecutive breeding seasons. All cytobrush samples were placed and stored in RNAlater and kept at -20° C until further processing. Uterine swabs were plated on sheep blood and MacConkey plates, incubated at 37° C in aerobic conditions and assessed for bacterial growth at 24, 48 and 72 hours. DNA was extracted from cytobrush samples using routine methods. The genomic DNArecovered was used as a template for PCR amplification of the V3–V4 variable region of the bacterial 16S rRNA gene. Amplicons were sequenced on the Illumina MiSeq platform. DNA sequencing data was then processed to assess the equine microbiome composition. The richness (number of bacterial species) and evenness (distribution of bacterial numbers) of bacterial genetic sequences was compared between maiden, barren and wet mares and correlated with reproductive success. The microbiome was highly dynamic and changed significantly between seasons within mares with a limited “core microbiome” being detected. In some mares, conventional culture techniques failed to identify known equine genital pathogens that were detected using 16S rRNA sequencing. While the richness and evenness of the microbiome was comparable in maiden, dry and wet mares, the overall bacterial abundance was higher in mares that were barren at the beginning of the season and failed to become pregnant during the season, when compared to successfully bred barren mares and all maiden mares. This was the first time the microbiome of the equine uterus was studied over several timepoints/seasons. Future studies could explore a prognostic test that allows prediction of fertility in barren mares. This work was funded by AgriFutures Australia.

55 Investigating the Potential Benefits of an Immunostimulant (Mycobacterium Cell Wall Fraction) Treatment on Increased Pregnancy Success of Embryo Transfer Recipients

Abstract Modulating the vaginal and uterine microbiome through the use of immunostimulants has the potential to improve fertility in cattle. The object of this study was to evaluate the potential benefits of an immunostimulant, mycobacterium cell wall fraction (MCWF), to increase pregnancy success of embryo transfer (ET) recipients. The three main objectives of this study were: determining if injecting ET recipient cows with MCWF increased proportion of cows with a corpus luteum (CL) on ET day, identifying differences in CL volume and progesterone concentration at the time of ET, and determining if ET recipients that received MCWF have greater pregnancy rate. A total of 926 beef cows at eight different locations were enrolled in this study. Primiparous and multiparous cattle were enrolled in a 7-day CO-Synch + CIDR estrous synchronization protocol. During this protocol, cows received an injection of GnRH (100-μg) at CIDR (controlled internal drug release) insertion [d -9], a prostaglandin F2α injection (25 mg), and CIDR removal [d-2] followed by another injection of GnRH (100 μg; d 0) within 60-66 hours after CIDR removal. At each location at (d 0), cows were sorted based on BCS and days post-partum and randomly assigned to one of two treatments: 1) MCWF- a single intramuscular injection (5 mL) of MCWF at (d 0; Amplimune, NovaVive Inc.; n = 452) or 2) Control- a single intramuscular injection (5 mL) of saline (n = 474). Seven days after (d 7), cows were examined by ultrasonography to determine if a CL was present. Recipient cows that had a CL received ET with a frozen or fresh in vivo or in vitro produced embryo in the ipsilateral uterine horn to the CL, with embryo type and grade balanced between treatments. Blood samples were collected in one location (n = 98) for analysis of plasma concentration of progesterone on (d 7) ET day. Pregnancy diagnosis was then performed by ultrasonography between 30 and 90 days of gestation. Data were analyzed using the GLIMMIX and MIXED procedures of SAS. Cow BCS and days postpartum were similar (P > 0.10) between treatments (5.5 ± 0.6 and 103 ± 14 days, respectively). Recipient cow utilization rate was similar between treatments (P = 0.49; 81.2 ± 0.42%). At the time of ET MCWF had larger CL than Control cows (5,768.6 ± 133.7 and 5,408.0 ± 128.5 mm3, respectively, P = 0.03). and increased progesterone concentration (2.68 ± 0.17 and 2.18 ± 0.14 ng/mL, respectively, P = 0.01). However, pregnancy rate to ET was similar (P = 0.922) between treatments (60.9 and 61.3 ± 0.05% for Control and MCWF, respectively). In conclusion, treatment of ET recipient cows with MCWF increased CL volume and concentration of progesterone at the time of ET but failed to generate greater pregnancy rates.

Challenging the Hypothesis of in Utero Microbiota Acquisition in Healthy Canine and Feline Pregnancies at Term: Preliminary Data.

At present, there are no data on the presence of bacteria in healthy canine and feline pregnancies at term. Here, we investigated the uterine microbiome in bitches (n = 5) and queens (n = 3) undergoing elective cesarean section in two facilities. Samples included swabs from the endometrium, amniotic fluid, and meconium, and environmental swabs of the surgical tray as controls. Culture and 16S rRNA gene sequencing were used to investigate the presence of bacteria. Culture was positive for 34.3% of samples (uterus n = 3, amniotic fluid n = 2, meconium n = 4, controls n = 0), mostly with low growth of common contaminant bacteria. With sequencing techniques, the bacterial abundance was significantly lower than in environmental controls (p < 0.05). Sequencing results showed a species-specific pattern, and significant differences between canine and feline bacterial populations were found at order, family, and genus level. No differences were found in alpha and beta diversities between feto-maternal tissues and controls (p > 0.05). Dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria in different proportions based on tissue and species. Culture and sequencing results suggest that the bacterial biomass is very low in healthy canine and feline pregnancies at term, that bacteria likely originate from contamination from the dam's skin, and that the presence of viable bacteria could not be confirmed most of the time.

178 Vaginal and uterine microbiome in metritic versus healthy dairy cows at disease diagnosis (day 7 to 14 postpartum), after clinical cure (day 28 to 35 postpartum) and at mid-lactation (day 80 to 165 postpartum)

178 Vaginal and uterine microbiome in metritic versus healthy dairy cows at disease diagnosis (day 7 to 14 postpartum), after clinical cure (day 28 to 35 postpartum) and at mid-lactation (day 80 to 165 postpartum)

Female reproductive tract microbiome and early miscarriages.

Miscarriage is one of the main causes of reproductive loss, which can lead to a number of physical and psychological complications and other long-term consequences. However, the role of vaginal and uterine microbiome in such complications is poorly understood. To review the published data on the function of the female reproductive tract microbiome in the pathogenesis of early miscarriages. The articles published over the past 20 years and deposited in PubMed, Google Academy, Scopus, Elibrary, ResearchGate, and EBSCO databases were analyzed. The review presents new data on the impact of the vaginal and uterine microbiome on the local immunity, including defense against sexually transmitted infections, and its association with other factors of miscarriages. The studies on the microbiome of non-pregnant women with recurrent miscarriages in the anamnesis, patients undergoing IVF, and pregnant women with miscarriages, as well as new directions in the microbiome research are discussed. The majority of studies have demonstrated that the dominant species of the vaginal and uterine microbiome in patients with early miscarriages are non-Lactobacillus bacteria. As many of these bacteria have not previously been detected by cultural studies and their role in obstetric complications is not well defined, further research on the female reproductive tract microbiome, including the microbiome of the cervix uteri, is needed to develop new approaches for the prognosis and prevention of miscarriages.

Studying the pre-implantation uterine microbiota in cattle using transabdominal laparoscopic low-volume lavage: Aiming for zero-contamination

Recent studies have suggested that bacteria associated with the female reproductive tract – the uterine microbiota – may be important for reproductive health and pregnancy success. Therefore, uterine microbiome research gained much interest in the last few years. However, it is challenging to study late postpartum uterine samples, since they hold a low microbial biomass. Next-generation sequencing techniques are very sensitive for microbial identification, but they cannot make a distinction between actual microbiota and contaminant bacteria or their DNA. Our aim was to test a new method to sample the bovine uterine lumen in vivo, while minimizing the risk of cross-contamination. In order to evaluate this method, we performed a descriptive assessment of the microbial composition of the obtained samples. Transabdominal, laparoscopic sampling of the uterine lumen was conducted in five Holstein-Friesian cows. Uterine fluid from the uterine horns was collected by low-volume lavage. DNA from the samples was extracted using two different DNA extraction methods, and negative controls (sampling blank controls and DNA extraction blank controls) were included. Bacteria were identified using 16S rRNA gene amplicon sequencing. In this proof-of-concept study, no evidence for authentically present uterine microbiota could be found. During laparoscopic sampling, some practical challenges were encountered, and the reliability of low-volume-lavage for the collection of a low microbial biomass could be questioned. By comparing two DNA extraction methods, a significant contamination background could be noticed originating from the DNA extraction kits.

Therapeutic intervention based on gene sequencing analysis of microbial 16S ribosomal RNA of the intrauterine microbiome improves pregnancy outcomes in IVF patients: a prospective cohort study.

A Lactobacillus-dominated microbiota in the endometrium was reported to be associated with favorable reproductive outcomes. We investigated in this study whether 16S ribosomal RNA (rRNA) gene sequencing analysis of the uterine microbiome improves pregnancy outcomes. This prospective cohort study recruited a total of 195 women with recurrent implantation failure (RIF) between March 2019 and April 2021 in our fertility center. Analysis of the endometrial microbiota by 16S rRNA gene sequencing was suggested for all patients who had three or more failed embryo transfers (ETs). One hundred and thirty-one patients underwent microbial 16S rRNA gene sequencing (study group) before additional transfers, while 64 patients proceeded to ET without that analysis (control group). The primary outcome was to compare the cumulative clinical pregnancy rate of two additional ETs. An endometrial microbiota considered abnormal was detected in 30 patients (22.9%). All but one of these 30 patients received antibiotics according to the bacterial genus detected in their sample, followed by treatment with probiotics. As a result, the cumulative clinical pregnancy rate (study group: 64.5% vs. control group: 33.3%, p = 0.005) and the ongoing pregnancy rate (study group: 48.9% vs. control group: 32.8%, p = 0.028) were significantly increased in the study group compared to the control group. Personalized treatment recommendations based on the microbial 16S rRNA gene sequencing of the uterine microbiota can improve IVF outcomes of patients with RIF. The University Hospital Medical Information Network (UMIN) Clinical Trial Registry: UMIN000036050 (date of registration: March 1, 2019).

Effects of Hydrosalpinx on Endometrial Receptivity and Uterine Microbiome: An Interesting Case of Double Uterus with Unilateral Hydrosalpinx

Endometrial receptivity array (ERA)—an objective tool used in assisted reproductive technology—is used for personalized embryo transfer in in vitro fertilization. Hydrosalpinx affects implantation through various mechanisms. However, its effects on ERA are not well established. In this case report, we present the diagnosis and treatment of a 34-year-old nulligravida woman with infertility for two years, obesity, double uterus with unilateral hydrosalpinx and right kidney deficiency. Based on ERA results, endometrial microbiome metagenomic analysis (EMMA), analysis of infectious chronic endometritis (ALICE), and CD138 immunostaining, the patient was treated with hormonal replacement cycle and amoxicillin/clavulanic acid. After one week of amoxicillin/clavulanic acid administration, the vitirified-warmed 4AA blastocyst was transferred to the left uterus—which was absent of hydrosalpinx and easily accessible to transfer and pregnancy was achieved. To the best of our knowledge, this case study is the first one in which we found that there were no differences between the left and right uterus in ERA, EMMA, ALICE, and CD138 immunostainings. Hence, we suggest that hydrosalpinx does not necessarily cause endometrial changes in all cases. Further research to evaluate the effects of hydrosalpinx on implantation with ERA and EMMA/ALICE is warranted.

PSIII-14 Reproductive Microbiome and Cytokine Profiles of Postpartum Beef Cows in Relation to Fertility

Abstract Shifts in commensal bacteria (i.e., Firmicutes) in the human microbiome are associated with compromised fertility. The objectives of this study were to characterize 1) the reproductive microbiome and 2) the cytokine concentrations prior to artificial insemination (AI) in vaginal and uterine flushes from postpartum beef cows. Twenty Bos indicus-influenced beef cows (~60 days postpartum and free of health issues) were weighed, body condition scored (BCS) then subjected to the Bee Synch II synchronization protocol on d-8 and timed AI on d0. Blood samples were collected on d-3, -1, 0, and 28. Vaginal and uterine flushes were collected on d-3 and d-1. On d28, pregnancy was determined by transrectal ultrasonography (Open, n=13 and Pregnant, n=7). Bacterial community analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. Cytokine analyses were conducted using the RayBiotech Quantibody® Bovine Cytokine Array Q1 kit per manufacturer's instructions. Weight, BCS, and vaginal bacterial communities by phyla were not different (P &amp;gt;0.05). In open cows, the uterine relative abundance of Firmicutes decreased between d-3 and -1 (60.4% ± 0.9% vs. 48.5% ± 3.2%, respectively; P&amp;lt; 0.05). In pregnant cows, the uterine relative abundance of Tenericutes significantly increased between d-3 and -1 (1.0% ± 0.1% vs. 7.6% ± 4.1%, respectively; P&amp;lt; 0.05). Additionally, the genus Ureaplasma tended to increase in relative abundance from d-3 to -1 (0.1% ± 0.1% vs. 7.3% ± 4.9%, respectively; P=0.06). Uterine concentrations of Interferon (IFN)-γ, Interleukin (IL)-1α and IL-21 were greater in open females compared to pregnant. Vaginal concentrations for IFN-α and chemokine ligand (CXCL)-10 were greater in open females compared to pregnant. Regardless of treatment, IL-13 increased from d-3 to d-1. These results suggest a distinct difference in the uterine microbiome and cytokine profiles prior to AI for resulting pregnant and open cows.

The healthy equine uterus harbors a distinct core microbiome plus a rich and diverse microbiome that varies with geographical location

The goal of this study was to understand the composition and existence of the resident uterine microbiome in healthy mares and to establish the presence of a core microbiome for the healthy equine uterus. We analyzed the microbiomes of 35 healthy mares that are long-time residents of three farms in Oklahoma, Louisiana, and Australia as well as that of 19 mares purchased from scattered owners in the Southern Mid-Western states of the United States. Over 6 million paired-end reads of the V4 region of the 16S rRNA gene were obtained resulting in 19,542 unique Amplicon Sequence Variants (ASVs). ASVs were assigned to 17 known phyla and 213 known genera. Most abundant genera across all animals were Pseudomonas (27%) followed by Lonsdalea (8%), Lactobacillus (7.5%), Escherichia/Shigella (4.5%), and Prevotella (3%). Oklahoma and Louisiana samples were dominated by Pseudomonas (75%). Lonsdalea (28%) was the most abundant genus in the Australian samples but was not found in any other region. Microbial diversity, richness, and evenness of the equine uterine microbiome is largely dependent on the geographical location of the animal. However, we observed a core uterine microbiome consisting of Lactobacillus, Escherichia/Shigella, Streptococcus, Blautia, Staphylococcus, Klebsiella, Acinetobacter, and Peptoanaerobacter.

Comparison of tumor microbiota between primary and recurrent or metastatic sites in women with advanced or relapsed endometrial and ovarian cancer (240)

Comparison of tumor microbiota between primary and recurrent or metastatic sites in women with advanced or relapsed endometrial and ovarian cancer (240)

Bacterial Communities of the Uterus and Rumen During Heifer Development With Protein Supplementation

Bacterial communities play major roles in rumen and uterine function toward optimal animal performance and may be affected by changes occurring during heifer development such as nutritional supplementation for optimal growth and the attainment of puberty. The effect of different levels of protein supplementation on ruminal and uterine bacterial communities following weaning was examined through first breeding of heifers. Angus heifers (n = 39) were blocked by initial body weight (BW) and randomly assigned to one of three 163-day (d) crude protein (CP) supplementation diets including control (10% CP, n = 14), 20% CP (n = 11), or 40% CP (n = 14) treatment groups. Growth and development were monitored by body weight, with blood progesterone concentration determined every 14 d to determine pubertal status. Uterine flush and rumen fluid were collected on d 56, 112, and 163 relative to the start of supplementation. Bacterial DNA was extracted from fluid samples, the V1–V3 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced then processed in R 4.1. Statistical analyses were performed in SAS 9.4 with a GLIMMIX procedure utilizing fixed effects of protein, month, pubertal status, and interactions, with random effects including BW, interaction of BW and protein, and heifer within the interaction, and repeated measures of day. In the uterus, pubertal status and day of supplementation affected the observed amplicon sequence variants (ASVs) and led to clustering of samples in a principal coordinate analysis (PCoA; P &amp;lt; 0.05), but no effect of protein supplementation was observed. Ruminal samples clustered in PCoA (P = 0.001), and observed ASVs were impacted over time (P &amp;lt; 0.0001), but no effect of protein supplementation was detected. In contrast, protein supplementation, pubertal status, and day of supplementation affected the abundance of multiple phyla and genera in the uterus and rumen (P &amp;lt; 0.05). Temporal and pubertal status effects on the heifer’s uterine bacterial communities potentially indicate a maturing uterine microbiome. Protein supplementation did not impact microbial diversity measures but did affect the abundance of individual bacterial phyla and genera that may provide future opportunities to manipulate bacterial community composition and maximize productivity.

P-384 Efficacy of endometrial microbiome metagenomic analysis with recurrent implantation failure and recurrent pregnancy loss: multicenter study in Japan

Abstract Study question Does the result of endometrial microbiome metagenomic analysis (EMMA), a next generation sequencing (NGS)-based test of the intrauterine microbiome, have an impact on pregnancy rate after the test? Summary answer After recommend treatment with antimicrobial and probiotic therapy, the group diagnosed as dysbiosis by EMMA achieved pregnancy significantly earlier than the group with Normal result. What is known already Using NGS technology, EMMA testing can determine the composition of the endometrial microbiome by analysing bacterial 16S ribosomal RNA with a focus on the lactobacillus population. Endometrial flora in patients undergoing in vitro fertilization (IVF) is often composed of pathogenic microorganisms which decrease implantation rates, such as the Enterobacteriaceae family, Staphylococcus spp., Escherichia coli and Gram-negative bacteria. Other studies also indicate that Lactobacillus spp. is a major microorganism in the endometrium. Especially, lactobacillus-dominated microbiota (LDM, defined as &amp;gt; 90% Lactobacillus spp.) in the endometrium has been reported to lead to better pregnancy outcomes than non-LDM (&amp;lt;90% Lactobacillus spp.). Study design, size, duration This study was a prospective, multicenter cohort study of 527 patients (under 42 years old) with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) at 14 IVF treatment facilities in Japan from June 2019 to August 2021. Defining RIF as three or more failed implantation attempts and RPL as two or more miscarriages, we examined the prognosis of two additional embryo transfers (ETs) after the EMMA test in patients who underwent the test. Participants/materials, setting, methods Endometrial tissue was obtained by aspiration from patients in day 15-25 of their menstrual cycles, and sample tissues were analyzed by NGS for EMMA. Participant centers treated patients according to the therapies specified in the reports, including antibiotic treatments, probiotic treatments, re-analysis, and embryo transfer. Multivariate analysis was performed using a generalized linear model with the endpoint of ongoing pregnancy. For the time-to-event analyses, we used Kaplan-Meier survival analysis to compare time to ongoing pregnancy. Main results and the role of chance The results of the first EMMA were as follows: 229 patients (43.4%) were normal with Lactobacillus spp. &amp;gt;90% (Normal group), 110 patients (20.9%) were abnormal with less than 90% Lactobacillus spp. and predominantly pathogenic bacteria (Abnormal group), and 188 patients (35.7%) were mild with low absolute amounts of bacteria and ultralow biomass indicating almost sterile (Mild + Ultralow group). There were no significant differences in background factors such as age, duration of infertility, number of previous ETs, or history of deliveries among the three groups. Gardnerella was the most pathogenic bacteria detected in patients with Abnormal EMMA results. All patients in the Abnormal group were treated with antimicrobials and probiotics, and those in Mild + Ultralow group were treated with probiotics. Odds ratio for ongoing pregnancy rate was 1.10(95%CI 0.67-1.82, p = 0.699) in Abnormal group and 1.23(95%CI 0.80-1.89, p = 0.342) in Mild + Ultralow group, respectively. After the intervention, ongoing pregnancies were comparable to those in Normal group.Analysis of time to pregnancy using Kaplan-Meier survival curves showed that Abnormal group had a significantly higher rate of ongoing pregnancies during the observation period than the other groups (p = 0.031). Limitations, reasons for caution Since this study was not necessarily limited to euploid embryos transferred after testing, an aging bias cannot be excluded. Since this study was conducted with all patients receiving EMMA, the effectiveness of the test needs to be further validated by comparison to patients without EMMA testing. Wider implications of the findings This study is the first multicenter study to demonstrate that the intervention based on EMMA reports improve pregnancy outcome in the patients with RIF and RPL. We suggest that the EMMA procedure, which aims at establishing an appropriate uterine microbiome, may be important for implantation and pregnancy continuation. Trial registration number UMIN000036917

Microbiome as a predictor of implantation.

Review the latest research on the female urogenital microbiome as a predictor of successful implantation. Lactobacillus crispatus seems to be beneficial species in a healthy female genital tract, although the presence of anaerobic bacteria and their impact has yet to be determined. The vaginal microbiome is associated with assisted reproductive technology (ART) outcome in terms of successful implantation and pregnancy. Approaches restoring a dysbiotic vaginal microbiome seem promising. It is questionable if a unique endometrial microbiome exists, given the low bacterial biomass, the invasiveness of endometrial sampling, and its associated high contamination risk. Future studies should focus on the whole microbiome using proteomics and metabolomics, as well as the virome to get a more holistic understanding of its role in reproduction. The vaginal and endometrial compartments are being studied to determine a healthy and unhealthy microbiome composition. Defining a healthy composition could provide insight into physiological processes related to the success of embryo implantation. The vaginal microbiome is easily accessible and its composition can be reliably assessed and can be associated with ART outcome. The existence of an endometrial or uterine microbiome is still debated, due to the combination of low biomass and unavoidable high risk of contamination during sampling.

Ligilactobacillus salivarius PS11610 exerts an effect on the microbial and immunological profile of couples suffering unknown infertility.

Unknown or idiopathic infertility has been associated with urogenital tract dysbiosis, reducing pregnancy and delivery ratios during assisted reproductive treatments (ART). The Ligilactobacillus salivarius PS11610 strain has shown extraordinary antimicrobial activity in vitro against urogenital pathogens as well as other probiotic characteristics. Therefore, an intervention study was performed to evaluate the effect of L. salivarius PS11610 on the microbial composition of urogenital tract in infertile couples with bacterial dysbiosis. Seventeen couples undergoing ART diagnosed with unknown infertility were selected. After confirming urogenital dysbiosis, they started a 6-month treatment with L. salivarius PS11610 (1 dose/12 h for female and 1 dose/24 h for male). Vaginal, seminal, glans, uterine and plasma samples were collected for determination of the microbiome and immune profile at the beginning and the end of the treatment. Supplementation with L. salivarius PS11610 significantly modified the urogenital microbiome composition in male and female samples, solving dysbiosis of 67% of the couples. Pathogens disappeared from the vaginal samples whereas Lactobacilli percentage increased after 3 and 6 months of treatment. Moreover, L. salivarius PS11610 changed the uterine microbiome that could be associated with a change of the uterine immune profile. Additionally, the probiotic intake could be associated with the observed change in the systemic immunological profile of couples. Finally, the pregnant and delivery ratio were improved. Probiotic supplementation with L. salivarius PS11610 improved the male and female urogenital tract microbiome, modulating the immune system and increasing pregnancy success in couples undergoing ART.