Endometrial Microbiome and Women’s Reproductive Health – Review of the Problem Endometrial Microbiome and Reproductive Health

Currently, unlike in the past, the endometrial cavity is not considered to be sterile. The endometrium is supposed to be dominated by Lactobacilli, but also their deficiency can be found in the reproductive tract of asymptomatic healthy women. Sometimes the endometrial microbiome is dominated by various pathological microorganisms, and this can lead to various conditions as chronic endometritis, chorioamnionitis and preterm birth. Their presence causes uterine inflammation and infection, release of pro-inflammatory molecules, uterine contractions, disruption of cervical barrier, premature rupture of membranes. Uterine dysbiosis is associated with recurrent implantation failure and recurrent miscarriages. As the microbiome is important for maintaining immunological homeostasis at the level of gastrointestinal tract Lactobacilli may play a similar function at the level of uterus. The lactobacillus-dominated uterine microbiome is of great importance for maintaining a hostile uterine microenvironment, embryo implantation, early pregnancy development and normal pregnancy outcome.

Characterization of an Ex Vivo Equine Endometrial Tissue Culture Model Using Next-Generation RNA-Sequencing Technology

Persistent mating-induced endometritis is a major cause of poor fertility rates in the mare. Endometritis can be investigated using an ex vivo equine endometrial explant system which measures uterine inflammation using prostaglandin F2α as a biomarker. However, this model has yet to undergo a wide-ranging assessment through transcriptomics. In this study, we assessed the transcriptomes of cultured endometrial explants and the optimal temporal window for their use. Endometrium harvested immediately post-mortem from native pony mares (n = 8) were sampled (0 h) and tissue explants were cultured for 24, 48 and 72 h. Tissues were stored in RNALater, total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were defined using DESeq2 (R/Bioconductor). Principal component analysis indicated that the greatest changes in expression occurred in the first 24 h of culture when compared to autologous biopsies at 0 h. Fewer DEGs were seen between 24 and 48 h of culture suggesting the system was more stable than during the first 24 h. No genes were differentially expressed between 48 and 72 h but the low number of background gene expression suggested that explant viability was compromised after 48 h. ESR1, MMP9, PTGS2, PMAIP1, TNF, GADD45B and SELE genes were used as biomarkers of endometrial function, cell death and inflammation across tissue culture timepoints. STRING assessments of gene ontology suggested that DEGs between 24 and 48 h were linked to inflammation, immune system, cellular processes, environmental information processing and signal transduction, with an upregulation of most biomarker genes at 24 h. Taken together our observations indicated that 24–48 h is the optimal temporal window when the explant model can be used, as explants restore microcirculation, perform wound healing and tackle inflammation during this period. This key observation will facilitate the appropriate use of this as a model for further research into the equine endometrium and potentially the progression of mating-induced endometritis to persistent inflammation between 24 and 48 h.

Regulatory Influence of Galanin and GALR1/GALR2 Receptors on Inflamed Uterus Contractility in Pigs

Uterine inflammation is a very common and serious pathology in domestic animals, the development and progression of which often result from disturbed myometrial contractility. We investigated the effect of inflammation on the protein expression of galanin (GAL) receptor subtypes (GALR)1 and GALR2 in myometrium and their role in the contractile amplitude and frequency of an inflamed gilt uterus. The gilts of the E. coli and SAL groups received E. coli suspension or saline in their uteri, respectively, and only laparotomy was performed (CON group). Eight days later, the E. coli group developed severe acute endometritis and lowered GALR1 protein expression in the myometrium. Compared to the pretreatment period, GAL (10−7 M) reduced the amplitude and frequency in myometrium and endometrium/myometrium of the CON and SAL groups, the amplitude in both stripes and frequency in endometrium/myometrium of the E. coli group. In this group, myometrial frequency after using GAL increased, and it was higher than in other groups. GALR2 antagonist diminished the decrease in amplitude in myometrium and the frequency in endometrium/myometrium (SAL, E. coli groups) induced by GAL (10−7 M). GALR1/GALR2 antagonist and GAL (10−7 M) reversed the decrease in amplitude and diminished the decrease in frequency in both examined stripes (CON, SAL groups), and diminished the drop in amplitude and abolished the rise in the frequency in the myometrium (E. coli group). In summary, the inflammation reduced GALR1 protein expression in pig myometrium, and GALR1 and GALR2 participated in the contractile regulation of an inflamed uterus.

Alterations in the Expression Profile of Serum miR-155, miR-223, miR-17, miR-200a, miR-205, as well as Levels of Interleukin 6, and Prostaglandins during Endometritis in Arabian Mares

So far the intimate link between serum microRNA (miRNA) and uterine inflammation in mares is unknown. We aimed (I) to investigate expression profile of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 (II) and to measure concentrations of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2) in serum of mares with healthy and abnormal uterine status (endometritis). This study was conducted on 80 Arabian mares: young (4–7 years), and old (8–14 years). Mares were divided into 48 sub-fertile (endometritis) and 32 fertile (control) at stud farms. Serum was collected for measuring IL-6, PGF2α, and PGE2, as well as miRNA isolation and qRT-PCR. Concentrations of IL-6, PGE2, and PGF2α were higher in mares with endometritis compared to control. Age of mares had a remarkable effect on IL-6, PGE2, and PGF2α concentrations. Relative abundance of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 was higher in both young and old mares with endometritis. We noticed that eca-miR-155, eca-miR-223, eca-miR-200a, and eca-miR-205 revealed higher expression level in old than young mares with endometritis. This is the first study that has revealed the changes in cell free miRNA and serum inflammatory mediators during endometritis, and these findings could be used for a better understanding the pathophysiology mechanisms of endometritis in equine.

Role of beta-adrenergic receptor subtypes in pig uterus contractility with inflammation

Uterine inflammation is a very common and serious condition in domestic animals. To development and progression of this pathology often lead disturbances in myometrial contractility. Participation of β1-, β2- and β3-adrenergic receptors (ARs) in noradrenaline (NA)-influenced contractility of the pig inflamed uterus was studied. The gilts of SAL- and E.coli-treated groups were administered saline or E.coli suspension into the uterine horns, respectively. Laparotomy was only done in the CON group. Compared to the period before NA administration, this neurotransmitter reduced the tension, amplitude and frequency in uterine strips of the CON and SAL groups. In the E.coli group, NA decreased the amplitude and frequency, and these parameters were lower than in other groups. In the CON, SAL and E.coli groups, β1- and β3-ARs antagonists in more cases did not significantly change and partly eliminated NA inhibitory effect on amplitude and frequency, as compared to NA action alone. In turn, β2-ARs antagonist completely abolished NA relaxatory effect on these parameters in three groups. Summarizing, NA decreases the contractile amplitude and frequency of pig inflamed uterus via all β-ARs subtypes, however, β2-ARs have the greatest importance. Given this, pharmacological modulation of particular β-ARs subtypes can be used to increase inflamed uterus contractility.

Effect of Kang Fu Yan capsule on phenol mucilage-induced intrauterine adhesion injury in female rats

Purpose: To investigate the effect of Kang fu yan capsule (KFYC) on phenol mucilage-induced intrauterine adhesion (IUA) in a rat model, and the underlying mechanisms. Methods: An IUA model was established by injecting 0.06 mL of 25 % phenol mucilage into the uterus of female Sprague-Dawley rats. The IUA model rats (n=59) were randomly divided into 5 groups: IUA group, fuke qianjin tablet group (FKQJT, 0.22 mg/kg), and 3 KFYC groups given different doses of the drug i.e. 0.13, 0.39and 1.17 mg/kg. A group of 10 healthy female rats served as control. After 19 days treatment, blood samples were collected for determination of IL-2 and IL-10 by ELISA, while uterine tissues were subjected to histological examination using hematoxylin and eosin staining (H&E) and Masson staining. Expressions of Notch1, recombination signal binding protein-JK (RBP-JK), a disintegrin and metalloprotease (ADAM)-12, ADAM-15, matrix metalloprotein-9 (MMP-9), and inhibitor of NF-κB (IĸB) in uterine tissues were determined using RT-qPCR and western blot analysis. Results: Compared to IUA group, histological results showed reduced inflammatory cell infiltration in rat uterine tissue of KFYC group. Moreover, KFYC significantly reversed uterine fibrosis (p < 0.05). Serum concentrations of IL-2 significantly decreased in KFYC groups (p < 0.05 or p < 0.01), and there was significant increases the serum concentrations of IL-10 in KFYC groups (p < 0.05 or < 0.01), when compared to IUA group. The mRNA and protein expressions of Notch1, RBP-JK, ADAM-12, ADAM-15, MMP-9 were also significantly down-regulated (p < 0.05), while protein expression of IĸB was upregulated in KFYC group, when compared to IUA group. Conclusion: KFYC exerts an anti-IUA effect via amelioration of uterine inflammation and fibrosis, probably via a mechanism involving regulation of Notch1/ADAM pathway.

Comparison of the Cytology Brush and Low Volume Lavage Techniques to Evaluate Endometrial Cytology for Diagnosing Endometritis in Chronically Infertile Mares

Endometritis is the most common cause of subfertility and infertility in mares that leads to great economical loss. Endometrial cytology (EC) is a reliable diagnostic tool, widely used to assess the health of the reproductive tract in both humans and domestic mammals. The aim of this study was to compare two techniques for the collection of EC samples in chronically infertile mares. Ten mares aged 4 to 20 were used. In each mare, samples for EC were collected first by cytology brush (CB) and then by low volume lavage (LVL) and stained using the Diff Quick stain. Each sample was assessed for the following parameters: background of the slides, quality and quantity of the cells obtained, number of inflammatory cells. Also, we assessed the number of operators, time and costs needed for each technique. More debris was noticed in the LVL samples, as well as a higher percentage of distorted cells. However, inflammatory cells were detected in all positive samples. Sampling by LVL required two to three operators and recovery of the lavage fluid was sometimes difficult. In conclusion, both techniques are accurate for the detection of uterine inflammation.

IL-1β mediates miR-34a promotes bovine endometritis via LGR4-NF-κB axis

Background Persistent endometritis lead by bacterial infections has lethal effects on the reproductive performance of dairy cattle, which not only compromise animal welfare but also delay or prevent pregnancy. MicroRNA (miRNA) miR-34 family plays a pivotal role in the inflammatory process; however, the precise mechanism of miR-34a in endometritis is still not thoroughly revealed. Methods In this study, we established bovine endometrial epithelial cell (BENDs) inflammation model and mouse model stimulation with Lipopolysaccharide (LPS) in vitro and in vivo. CCK-8 was used to assess cell viability. H&E was used to characterize morphology. Immunohistochemistry, immunofluorescence, qRT-PCR and western blot assays were performed to measure the mRNA or protein expression of related genes. Online database, plasmid construction and dual-luciferase Reporter gene assays were applied to predict and validate the interaction between miR-34a and its target gene LGR4, and mice were injected vaginally with local antagomir to validate the role of miR-34a in murine uterine inflammation. Results Here, we report that miR-34a suppresses LGR4 gene expression by targeting its 3'untranslated regions (3'UTR). The miR-34a was up-regulated in cow uterine tissues and bovine endometrial epithelial cell (BENDs) stimulation with LPS. It further induces the release of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α by activating the phosphorylation level of NF-κB p65. Furthermore, we also revealed that IL-1β was responsible for the upregulation of miR-34a transcription and downregulation of LGR4 in an IL-1β-dependent manner. Conclusions Taken together, our study confirmed that miR-34a is regulated by IL-1β and suppress the level of LGR4 3’UTR which in turn exacerbates the inflammatory response. Thus knockdown miR-34a might be a new indirection for treatment endometritis.

Serum miR-155, miR-223, miR-17, miR-200a, miR-205, Interleukin 6, and Prostaglandins as Novel Diagnostic Markers for Endometritis in Arabian Mares

Background: So far the intimate link between serum microRNA (miRNA) and uterine inflammation in mares is unknown. We aimed (I) to investigate the expression profile of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 (II) and to measure the concentrations of interleukin 6 (IL-6), and prostaglandins (PGF2α& PGE2) in serum of Arabian mares with healthy and abnormal uterine status (endometritis).Methods and Results: This study was conducted on 80 Arabian mares; young (4-7 years), and old (8-14 years). These animals were divided into 48 sub-fertile including 16 young and 32 old mares suspected of endometritis and 32 fertile as control (24 young and 8 old) at stud farms. Serum samples were collected for measuring IL-6, PGF2α, and PGE2 concentrations, as well as serum miRNA isolation and qRT-PCR. Serum concentrations of IL-6, PGE2, and PGF2α were higher (P≤0.001) in mares with endometritis (young and old) compared to the control ones. Age of mares had a remarkable effect(0.001≤P≤0.01) onIL-6, PGE2, and PGF2αconcentrations. The relative abundance of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 was higher (P≤0.001) in both young and old mares with endometritis. We noticed that eca-miR-155, eca-miR-223, eca-miR-200a, and eca-miR-205 revealed higher (0.001≤P≤0.01) expression level in old than young mares with endometritis. Conclusions: To the best of our knowledge, this is the first study revealed that serum miRNA and serum inflammatory mediators (IL-6, PGE2, and PGF2α) could be used as non-invasive gold standard biomarkers, and therefore might be served as an important additional diagnostic tool for endometritis in Arabian mares.

Hydrogen Gas: A Novel Type of Antioxidant in Modulating Sexual Organs Homeostasis

Sex is a science of cutting edge but bathed in mystery. Coitus or sexual intercourse, which is at the core of sexual activities, requires healthy and functioning vessels to supply the pelvic region, thus contributing to clitoris erection and vaginal lubrication in female and penile erection in male. It is well known that nitric oxide (NO) is the main gas mediator of penile and clitoris erection. In addition, the lightest and diffusible gas molecule hydrogen (H2) has been shown to improve erectile dysfunction (ED), testis injuries, sperm motility in male, preserve ovarian function, protect against uterine inflammation, preeclampsia, and breast cancer in female. Mechanistically, H2 has strong abilities to attenuate excessive oxidative stress by selectively reducing cytotoxic oxygen radicals, modulate immunity and inflammation, and inhibit injuries-induced cell death. Therefore, H2 is a novel bioactive gas molecule involved in modulating sexual organs homeostasis.