Uterine Immunity Research Articles

Harnessing Vaginal Probiotics for Enhanced Management of Uterine Disease and Reproductive Performance in Dairy Cows: A Conceptual Review.

Uterine disease in cattle impairs reproductive performance and profitability and increases antibiotic use and antimicrobial resistance. Thus, probiotics offer a promising alternative therapy. This review presents conceptual findings on the efficacy of probiotics in managing uterine diseases and fertility in cows. Probiotics containing Lactobacillus spp. and Bifidobacterium spp. individually or as composite formulations are known to improve fertility. Strategic intravaginal administration of these formulations would likely enhance uterine immunity, particularly during the postpartum period. While current findings on the benefits to uterine health are encouraging, there is still significant knowledge missing, including a lack of empirical information from large-scale field trials. This review underscores the need for evidence-based guidelines for probiotics, such as genomic selection of formulations, targeted delivery, or potential synergy with other interventions. Future research should address these gaps to maximize the potential of probiotics in managing uterine diseases and enhancing the reproductive health of dairy cattle.

Activation of sperm Toll-like receptor 2 induces hyperactivation to enhance the penetration to mucus and uterine glands: a trigger for the uterine inflammatory cascade in cattle.

It is known that sperm and seminal plasma (SP) affect uterine immunity. In cattle, artificial insemination enables breeding by depositing frozen and largely diluted sperm with a negligible amount of SP into the uterus. Thus, the present study focused on the impact of frozen-thawed sperm on bovine uterine immunity. We have previously shown that in the bovine uterus, sperm swim smoothly over the luminal epithelium and some sperm interact with uterine glands to induce a weak inflammatory response mainly via the endometrial Toll-like receptor 2 (TLR2) signaling. However, the process by which sperm is encountered in the uterine glands is not completely clear. The present study intended to evaluate the role of sperm-TLR2 in sperm-uterine mucus penetration for reaching the glandular epithelium to induce the uterine immune response. To activate and block sperm-TLR2, they were treated with TLR2 agonist and antagonist, respectively. TLR2 activation enhanced sperm hyperactivation and improved its capacity to penetrate the artificial viscoelastic fluid and estrous-uterine-mucus. In contrast, TLR2-blocked sperm showed completely opposite effects. It is noteworthy, that the TLR2-activated sperm that penetrated the uterine mucus exhibited increased motile activity with hyperactivation. In the sperm-endometrial ex-vivo model, a greater amount of TLR2-activated sperm entered the uterine glands with an immune response, which was seen as the upregulation of mRNA expression for TNFA, IL1B, IL8, PGES, and TLR2 similar to those in control sperm. On the other hand, a lesser amount of TLR2-blocked sperm entered the uterine glands and weakened the sperm-induced increase only in PGES, suggesting that penetration of a certain number of sperm in the uterine gland is necessary enough to trigger the inflammatory response. Altogether, the present findings indicate that activation of sperm-TLR2 promotes their hyperactivation and mucus penetration with greater motility, allowing them to enter into the uterine glands more. This further suggests that the hyperactivated sperm contributes to triggering the pro-inflammatory cascade partly via TLR2 in the uterus.

O-031 Activation of NLRP3 inflammasome enhances neutrophil extracellular traps in women with stage III/IV endometriosis: convergence of multiple signaling portray association between genetics and lesion type

Abstract Study question Do NOD-like receptor pyrin-containing 3 (NLRP3) signaling affect neutrophil extracellular traps (NET)-osis in women with stage III/IV endometriosis? Summary answer Inflammasome activation whether due to pathogen (disease itself) or damage-associated molecular pattern (through NET) might be one mechanism involved in establishing perturbed endometrium in endometriosis. What is known already Endometriosis is characterized by number of processes like vascularization, hypernociception, and fibrosis, cardinal cause being inflammation. Recently, expression of inflammasome components, including NLRP3 and apoptosis signal-regulating kinase has been demonstrated in human endometrium cueing involvement in uterine innate immunity. Moreover, a novel extracellular killing mechanism, NET, is documented to reflect an inflammatory status in deep infiltrating endometriosis. Since, endometriosis demonstrates similarities with chronic inflammatory and autoimmune disorders; we postulated inflammatory responses in endometriosis may become modulated through a feed-forward loop of NET-induced specific cytokine production thus providing insights against potentiality of endometriotic cells to limit progression of the disease. Study design, size, duration Twenty-two consented women (24-39 years) with endometriosis (Group A) (Stages III–IV) (based on ASRM-2018 guidelines) and age-matched counterpart/s of male sub-fertility (Group B; control), free of uterine abnormalities (n = 18) were recruited between January to December 2021 from Institute of Reproductive Medicine, Kolkata. Eutopic endometrium, were collected from women undergoing diagnostic laparoscopy (Group A) or by curettage from women undergoing endometrial ablation (Group B). Serum was collected during window-of-implantation (LH + 7) for both the group/s. Participants/materials, setting, methods Pro-(IFN-g, TNF-a, IL-6, TGF-b) and anti-inflammatory (IL-10, IL-13, IL-4, IL-5) cytokines was evaluated by western blot. Expression level of inflammasome-related proteins (NLRP3, ASC, CASPASE1, PYCARD, IL-1-b) and mRNA expression was estimated by western-blot and quantitative-real-time PCR (qRT-PCR) respectively from tissue biopsies in Group A and B. SYTOX® green assay by flow-cytometry and neutrophil-elastase activity by immunofluorescence was done to quantify and characterize NET production in blood monocytes. P < 0.05 was considered statistically significant. Main results and the role of chance The mean (±SD) age of study population was 31.6±5.2 years. Hyperestrogenic milieu possibly stimulated (p < 0.001) pro-inflammatory molecules (IFN-g, TNF-a, IL-6, TGF-b) in endometriosis as observed by western-blot and qRT-PCR. A significant up-regulation (p < 0.001) was observed in relative mRNA expression of NLRP3 and PYCARD gene in Group A. However, caspase 1 expression documented non-significant variation in biopsies from endometriosis patients. Western blot corroborated the finding/s. The outcome of NLRP3 activation was supported by increased (p < 0.002) mRNA expression of IL-1b. NETs were detected significantly higher (p < 0.01) in 54.54% (12/22) patients in group A compared to control (16.66%; (3/18)). Moreover, quantification of NETs showed a significantly higher amount in endometriosis compared to group B (0.097 vs. 0.02, p < 0.03). Spermann-rank correlation by SPSS version22 revealed positive correlation between IL-1b with NLRP3 (r = 0.56, p< 0.001), PYCARD (r = 0.11, p< 0.01); and caspase1 (r = 0.16, p< 0.01) and IL-1b (r = 0.47, p< 0.01) with NET-positive cell/s in endometriosis. Moreover, posterior cul-de-sac lesions correlated positively with NET-positive cell/s (r = 0.29; p < 0.01) cueing to possible prognostic marker/s. In summary, a coordinated-fashioned action of components of NLRP3 inflammasome machinery may regulate production of NET/s forming a complex network allowing communication between cell-types in order to maintain viability and development of endometrial lesion/s. Limitations, reasons for caution Our findings need to be replicated in larger study cohort/s, especially stratified by severity of endometriosis sub-groups and in women of different ethnicities. Another limitation is the missing information about NETs in normal functioning human endometrium throughout the menstrual cycle. Wider implications of the findings The proposed study aims to understand potential role of NLRP3 inflammasome complex as a “double-edged sword” in the development and pathophysiology of endometriosis. Therapeutic alternatives that aim to re-balance the pro- to anti-inflammatory milieu in the endometrium should consider the inflammasome as part of the equation. Trial registration number Not applicable

Uterine Natural Killer Cells: A Rising Star in Human Pregnancy Regulation.

Uterine natural killer (uNK) cells are an immune subset located in the uterus. uNK cells have distinct tissue-specific characteristics compared to their counterparts in peripheral blood and lymphoid organs. Based on their location and the pregnancy status of the host, uNK cells are classified as endometrial NK (eNK) cells or decidua NK (dNK) cells. uNK cells are important in protecting the host from pathogen invasion and contribute to a series of physiological processes that affect successful pregnancy, including uterine spiral artery remodeling, fetal development, and immunity tolerance. Abnormal alterations in uNK cell numbers and/or impaired function may cause pregnancy complications, such as recurrent miscarriage, preeclampsia, or even infertility. In this review, we introduce recent advances in human uNK cell research under normal physiological or pathological conditions, and summarize their unique influences on the process of pregnancy complications or uterine diseases. Finally, we propose the potential clinical use of uNK cells as a novel cellular immunotherapeutic approach for reproductive disorders.

A combination of immune cell types identified through ensemble machine learning strategy detects altered profile in recurrent pregnancy loss: a pilot study

To compare the immunologic profiles of peripheral and menstrual blood (MB) of women who experience recurrent pregnancy loss and women without pregnancy complications. Explorative case-control study. Cross-sectional assessment of flow cytometry-derived immunologic profiles. Academic medical center. Women who experienced more than 2 consecutive miscarriages. None. Flow cytometry-based immune profiles of uterine and systemic immunity (recurrent pregnancy loss, n = 18; control, n = 14) assessed by machine learning classifiers in an ensemble strategy, followed by recursive feature selection. In peripheral blood, the combination of 4 cell types (nonswitched memory B cells, CD8+ T cells, CD56bright CD16- natural killer [NKbright] cells, and CD4+ effector T cells) classified samples correctly to their respective cohort. The identified classifying cell types in peripheral blood differed from the results observed in MB, where a combination of 6 cell types (Ki67+CD8+ T cells, (Human leukocyte antigen-DR+) regulatory T cells, CD27+ B cells, NKbright cells, regulatory T cells, and CD24HiCD38Hi B cells) plus age allowed for assigning samples correctly to their respective cohort. Based on the combination of these features, the average area under the curve of a receiver operating characteristic curve and the associated accuracy were >0.8 for both sample sources. A combination of immune subsets for cohort classification allows for robust identification of immune parameters with possible diagnostic value. The noninvasive source of MB holds several opportunities to assess and monitor reproductive health.

Pathogenic Infections during Pregnancy and the Consequences for Fetal Brain Development.

Pathogens comprised of viruses, bacteria, gut microbiome, and parasites are a leading cause of ever-emerging diseases in humans. Studying pathogens for their ability to cause diseases is a topic of critical discussion among scientists and pharmaceutical centers for effective drug development that diagnose, treat, and prevent infection-associated disorders. Pathogens impact health either directly by invading the host or by eliciting an acute inflammatory immune response. This paradigm of inflammatory immune responses is even more consequential in people who may be immunocompromised. In this regard, pregnancy offers an altered immunity scenario, which may allow the onset of severe diseases. Viruses, such as Influenza, HIV, and now SARS-CoV-2, associated with the COVID-19 pandemic, raise new concerns for maternal and fetal/neonatal health. Intrauterine bacterial and parasitic infections are also known to impact pregnancy outcomes and neonatal health. More importantly, viral and bacterial infections during pregnancy have been identified as a common contributor to fetal brain development defects. Infection-mediated inflammatory uterine immune milieu is thought to be the main trigger for causing poor fetal brain development, resulting in long-term cognitive impairments. The concept of in utero programming of childhood and adult disorders has revolutionized the field of neurodevelopment and its associated complications. Recent findings in mice and humans clearly support the idea that uterine immunity during pregnancy controls the health trajectory of the child and considerably influences the cognitive function and mental health. In this review, we focus on the in utero programming of autism spectrum disorders (ASD) and assess the effects of pathogens on the onset of ASD-like symptoms.

Immunotoxicity and uterine transcriptome analysis of the effect of zearalenone (ZEA) in sows during the embryo attachment period

Zearalenone is a mycotoxin and a pollutant that is commonly found in crops. Once ingested, ZEA can cause disturbances in the immune system and produce immunotoxicity. However, there is little research on the effect of ZEA exposure on the relationship between immune regulation and embryo implantation in the uteri of sows. Embryo implantation relies upon the fact that the relationship between the maternal and fetal immune systems is balanced. This balance is provided by the joint regulation of immune organs, cytokines, and uterine immunity. In this study, we investigated 20 sows with an initial weight of 100.00 ± 5.00 kg and 200 days in age. The sows were fed with diets containing ZEA at concentrations of 0 mg/kg, 1 mg/kg, 2 mg/kg, and 10 mg/kg, respectively, from 8 to 14 days of gestation. We studied immunotoxicity and the uterine transcriptomics associated with the effect of ZEA in sows during embryo attachment. Following ZEA treatment, serum biochemical analysis and RT-qPCR were used to detect the concentration and mRNA expression levels of immunoglobulin IgA, IgG, and IgM, in the serum and spleen, respectively. The same analysis was carried out for a range of cytokines in the serum and spleen: IL-1, IL-2, IL-6, IL-10, and TNF. Uterine transcriptome analysis revealed 75, 215, and 81 genes that were differentially expressed in the 0 mg/kg vs 1 mg/kg treatment, 0 mg/kg vs 10 mg/kg treatment, and 1 mg/kg vs 10 mg/kg treatment, respectively. GO terms analysis showed that the up-regulated genes related to the immune system were highly expressed. KEGG pathway analysis further revealed the importance of several metabolic pathways, including drug metabolism-cytochrome P450, the cytokine-cytokine receptor interaction pathway, and calcium signaling pathways. The differentially expressed genes were confirmed by quantitative real-time PCR. These findings expand our understanding of the gene expression profiles and signaling pathways associated with the immune response to ZEA exposure in sows during the embryo implantation window. This study provides valuable information for clarifying the molecular mechanism of ZEA's immunotoxicity to early pregnant sows in the future.

P18: Programmed expression of transthyretin (TTR) during normal pregnancy and effects of dysregulated TTR on uterine immunity and pregnancy in mice

P18: Programmed expression of transthyretin (TTR) during normal pregnancy and effects of dysregulated TTR on uterine immunity and pregnancy in mice

In utero immune programming of autism spectrum disorder (ASD)

Maladaptation of immune tolerance at the maternal-fetal interface affects balanced maternal-fetal cross-talk and placental health and is associated with adverse pregnancy outcomes. The concept of in utero programming of childhood and adulthood diseases has revolutionized the research on the role of pregnancy in maternal, neonatal, and adult health. However, it is not yet well understood whether dysregulation of uterine immunity contributes to any health consequences during childhood or later in life. Recent observations in mice and humans have strongly supported the notion that uterine immunity during pregnancy determines the health trajectory of the offspring and significantly impacts cognitive function and mental health. Importantly, IL-17a producing Th17 T cells have been projected as the main contributors to heterogeneous pathological and behavioral phenotypes associated with autism spectrum disorder (ASD). However, since normal pregnancy is associated with little or no Th17 cells at the maternal-fetal interface, it is not clear how and when the Th17 T cells are generated and which interventions can ameliorate the ASD-like features in newborns. We propose that infection-associated uterine immune activation within a critical window of development may propel trans-differentiation of Th17 T cells that eventually affect fetal brain development and induce ASD-like behavioral phenotype in the offspring.

Selected Uterine Immune Events Associated With the Establishment of Pregnancy in the Dog.

In the dog, implantation takes place at approximately 17 days of embryonal life and, while exposed to relatively high circulating progesterone concentrations, embryos presence is required for the formation of decidua. Furthermore, a balance between pro- and anti-inflammatory responses in conceptus-maternal communication is crucial for the onset of pregnancy. Strikingly, the understanding of such immune mechanisms in canine reproduction is still elusive. Here, canine uterine samples from pre-implantation (day 10–12, E+) and corresponding non-pregnant controls (E–), implantation (day 17, Imp) and post-implantation (day 18–25, Post-Imp) stages of pregnancy were used to investigate the expression and localization of several immune-related factors. The most important findings indicate increased availability of CD4, MHCII, NCR1, IDO1, AIF1, CD25, CCR7, and IL6 in response to embryo presence (E+), while FoxP3 and CCL3 were more abundant in E– samples. Implantation was characterized by upregulated levels of FoxP3, IL12a, ENG, and CDH1, whereas CD4, CCR7, IL8, and -10 were less represented. Following implantation, decreased transcript levels of TNFR1, MHCII, NCR1, TLR4, CD206, FoxP3, and IL12a were observed concomitantly with the highest expression of IL6 and IL1β. MHCII, CD86, CD206, CD163, TNFα, IDO1, and AIF1 were immunolocalized in macrophages, CD4 and Nkp46 in lymphocytes, and some signals of IDO1, AIF1, and TNF-receptors could also be identified in endothelial cells and/or uterine glands. Cumulatively, new insights regarding uterine immunity in the peri-implantation period are provided, with apparent moderated pro-inflammatory signals prevailing during pre-implantation, while implantation and early trophoblast invasion appear to be associated with immunomodulatory and rather anti-inflammatory conditions.

Novel predictive and therapeutic options for better pregnancy outcome in frozen embryo transfer cycles.

Since 1978, in the first decades of in vitro fertilization (IVF), the use of ovarian hyperstimulation allowed for the development and transfer of multiple embryos. As IVF technology improved, the number of multiple pregnancies increased, which led to gradual reduction in the number of embryos that were transferred. Embryo freezing (vitrification) was recommended to allow subsequent transfer if the fresh cycle was unsuccessful. However, experimentation has continued to improve pregnancy outcomes. We discuss here the significance of frozen embryo transfer cycle and the impact of uterine and peripheral immunity dominated by NK cells and regulatory T cells and human chorionic gonadotropin on pregnancy outcome in this innovative mode of IVF therapy.

Bovine Viral Diarrhoea Virus Infection Disrupts Uterine Interferon Stimulated Gene Regulatory Pathways During Pregnancy Recognition in Cows.

In cattle, conceptus-derived interferon tau (IFNT) is the pregnancy recognition (PR) signal. Our previous studies showed that non-cytopathic bovine viral diarrhoea virus (ncpBVDV) infection inhibited IFNT-induced interferon stimulated gene (ISG) expression, potentially causing early embryonic death. This study investigated the effect of bovine viral diarrhoea virus (BVDV) infection on upstream regulatory pathways of ISG production using an established PR model. Uterine endometrial cells from 10 apparently healthy and BVDV free cows were cultured and treated with 0 or 100 ng/mL IFNT for 24 h in the presence or absence of ncpBVDV infection. Microarray and pathway analysis were used to determine the IFNT-induced upstream regulators. Expression of the genes associated with the identified pathways were quantified with qPCR. IFNT challenge activated the signalling pathways associated with IFN receptors, JAK1/TYK2, IRFs and STATs and ncpBVDV infection inhibited the activation of IFNT on this pathway. Inhibition of this upstream signalling pathway may thus reduce ISG production to disrupt maternal PR. In addition, the reduction of uterine immunity by ncpBVDV infection may predispose the animals to uterine infection, which in turn impairs their reproductive performance. This provides a mechanism of how BVDV infection leads to early pregnancy failure in cows.

Uterine inflammatory conditions in cows: Types and etiopathogenesis

Uterine inflammatory conditions with associated fertility disorders are still a serious problem in dairy cow breeding, causing significant economic losses for breeders. Losses result from the costs of uterine infection treatment, fertility disorders, low pregnancy rates, reduced production of milk and the culling of the best cows in the herd due to infertility. According to literature data, uterine inflammatory conditions in cows are closely connected with the postpartum period, and reproductive system infections that occur at this time may persist beyond the postpartum period and cause reduced fertility in cows. Pathogenic microbiota are an underlying cause of the development and persistence of uterine inflammatory conditions. The microorganisms with an affinity for the endometrium that are most frequently isolated from the uterus of cows with metritis and endometritis are Trueperella pyogenes, Escherichia coli, Fusobacterium necrophorum and Prevotella melaninogenicus. No infectious agents may be detected in some inflammatory conditions that are related to the disregulation of intracellular prostaglandin synthesis in uterine endometrial cells. This condition, which is due to infection, may persist for differing periods of time after the elimination of pathogens and may impair the fertility of cows. It is currently believed that the persistence of inflammatory conditions of the uterus depends on the level of anti-infective immunological mechanisms, which not only eliminate pathogens and damaged tissue, but also protect the endometrium thanks to their regulatory properties. Not all defence mechanisms in the uterus have been discovered. Phagocytosis is the first and most important mechanism of defence against pathogens in the uterus of cows, involving specialized phagocytic cells, mainly neutrophils, monocytes and macrophages, which belong to the innate immune mechanisms, as well as migrate from blood at the inflammation site as a specific response. Immune system cells and endometrial epithelial cells, which until recently have been believed to be only a passive barrier to microorganisms, are actively involved in the recognition and initiation of the antiinfective immune response. According to many authors, disorders of the local mechanisms of uterine immunity are a basic cause of metritis and endometritis in dairy cows.

Selected leukocyte subpopulations in peripheral blood and uterine washings in cows before and after intrauterine administration of cefapirin and methisoprinol.

The aim of the study was to evaluate the selected lymphocyte subpopulations TCD4, TCD8, BCD21, BCD25, CD18, CD11b, and MHC II in blood and uterine flush of cows with endometritis, before and after intrauterine (i.u.) administration of cefapirin and methisoprinol. The research was carried out on 28 cows with clinical endometritis. Animals were divided into four groups, each composed of seven cows, depending on the i.u. preparation used: Group A, cefapirin; Group B, methisoprinol; Group C, cefapirin and methisoprinol simultaneously; and a control group-without medication. The study was performed using flow cytometry method. Summarizing the results of the research, i.u. infusion of cefapirin caused a weakening of the effector phase of the local uterine immune response; however, it enhanced leukocyte chemotaxis and antigen presentation. After i.u. administration of methisoprinol, the stimulation of specific uterine immunity mechanisms was mainly observed. The use of both mentioned preparations showed the strengthening of specific uterine immunological mechanisms presumably caused by methisoprinol, despite the inhibitory effect of the antibiotic. Intrauterine use of immunostimulatory substances can improve the effectiveness of the endometritis treatment in cows by improving specific local mechanisms of uterine immunity. As a consequence, it may enhance the effector function of immune competent cells and finally eliminate inflammation.

P32: Gestational age‐dependent expression of Transthyretin and its effects on uterine immunity and pregnancy in WT and human TTR transgenic mice

American Journal of Reproductive ImmunologyVolume 81, Issue S1 p. 53-53 ABSTRACTS P32: Gestational age-dependent expression of Transthyretin and its effects on uterine immunity and pregnancy in WT and human TTR transgenic mice First published: 31 May 2019 https://doi.org/10.1111/aji.31_13120Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume81, IssueS1Special Issue: The 39th Annual Meeting of the American Society for Reproductive Immunology, 12-15 June 2019, Grand Rapids, Michigan, USAMay 2019Pages 53-53 RelatedInformation

Uterine infection and immunity in cattle

Uterine infection and immunity in cattle

Improved uterine immune mediators in Holstein cows supplemented with rumen-protected methionine and discovery of neutrophil extracellular traps (NET)

During the transition from prepartum to early lactation, dairy cows often experience negative energy balance (NEB) that may result in reproductive stress and decreased fertility. The objective of this study was to observe the effects of rumen-protected methionine (RPM) on plasma amino acid concentrations, uterine cytology, immunohistochemistry (IHC) of glutathione peroxidase 1 (GPX) and superoxide dismutase 1 (SOD), and to confirm neutrophil extracellular trap (NET) formation. Multiparous Holstein cows (n = 20) were randomly assigned to two treatments starting at 21 d before calving until 73 days in milk (DIM). Treatments were: CON (n = 9, no supplementation, TMR with a Lys:Met = 3.5:1) and MET (n = 11, TMR + Smartamine® M with a Lys:Met = 2.8:1). Uterine endometrial biopsies, uterine cytology, and blood samples from the coccygeal artery or vein were collected at 15, 30, and 73 DIM. Blood plasma samples were analyzed for amino acids and metabolites. Uterine biopsies were analyzed for NET formation, neutrophil numbers, as well as GPX and SOD by IHC. Additionally, uterine cytology was analyzed for polymorphonuclear neutrophil (PMN) to epithelial cell percentage. Cows in CON had lower methionine plasma concentrations (18.05 ± 2.0 μM) than cows in MET (30.39 ± 1.6 μM). Cows in CON had greater cystine plasma concentrations (3.62 ± 0.3 μM) than cows in MET (2.8 ± 0.3 μM). No treatment differences were observed for SOD or GPX in the endometrium. Cows in CON tended to have a high score for positively immunolabeled GPX cells at 15 DIM than cows in MET. No treatment differences were observed for the percentage of PMN in uterine cytology, number of neutrophils, or extent of NET formation in the endometrium. A treatment by time interaction was observed for PMN percentage and the number of neutrophils: cows in MET tended to have greater PMN percentages than cows in CON at 15 DIM which decreased for subsequent days and cows in MET had greater neutrophil numbers in the endometrium at 30 DIM than cows in CON. In conclusion, dietary supplementation of RPM altered plasma amino acid concentrations and increased neutrophil infiltration in the postpartum period, suggesting improved uterine immunity.

Acute bovine viral diarrhea virus infection inhibits expression of interferon tau-stimulated genes in bovine endometrium.

Bovine viral diarrhea virus (BVDV) can evade host detection by downregulation of interferon signaling pathways. Infection of cows with noncytopathic (ncp) BVDV can cause early embryonic mortality. Upregulation of type I interferon stimulated genes (ISGs) by blastocyst-secreted interferon tau (IFNT) is a crucial component of the maternal recognition of pregnancy (MRP) in ruminants. This study investigated the potential of acute BVDV infection to disrupt MRP by modulating endometrial ISG expression. Endometrial cells from 10 BVDV-free cows were cultured and treated with 0 or 100 ng/ml IFNT for 24 h in the absence or presence of ncpBVDV infection to yield four treatment groups: CONT, ncpBVDV, IFNT, or ncpBVDV+IFNT. ncpBVDV infection alone only upregulated TRIM56, but reduced mRNA expression of ISG15, MX2, BST2, and the proinflammatory cytokine IL1B. As anticipated, IFNT treatment alone significantly increased expression of all 17 ISGs tested. In contrast to the limited effect of ncpBVDV alone, the virus markedly inhibited IFNT-stimulated expression of 15 ISGs tested (ISG15, HERC5, USP18, DDX58, IFIH1, IFIT1, IFIT3, BST2, MX1, MX2, RSAD2, OAS1Y, SAMD9, GBP4, and PLAC8), together with ISG15 secreted protein. Only TRIM56 and IFI27 expression was unaltered. IL1B expression was reduced by the combined treatment. These results indicate that acute ncpBVDV infection may decrease uterine immunity and lead to MRP failure through inhibition of IFNT-stimulated endometrial ISG production. This in turn could reduce fertility and predispose cows to uterine disease, while evasion of the normal uterine immune response by ncpBVDV may contribute to maintenance and spreading of this economically important disease.

Transcriptional abundance of antioxidant enzymes in endometrium and their circulating levels in Zebu cows with and without uterine infection

Oxidative stress during peripartum period may compromise the uterine immunity. In the present study, we assessed the oxidative stress and antioxidant status during peripartum period and studied their relationship with postpartum uterine infection in dairy cows. Peripheral blood concentrations of total antioxidant capacity (TAC), malondialdehyde (MDA) and nitric oxide (NO) were determined (day −21, −7, on the day of calving and day +7, +21, +35) in normal (n=11), puerperal metritic (n=7) and clinical endometritic (n=6) cows. Endometrial biopsy was performed on the day of calving and expression of CAT, GPx4 and SOD2 genes was studied using qRT-PCR. Puerperal metritic cows had significantly (P<0.05) lower TAC (on day −7, day 0, day +7, +21 & +35), higher MDA (on day −21, −7 & on the day of calving) and NO (on day 0, +7 & day +35) concentrations compared to normal cows. Similarly, clinical endometritic cows had significantly (P<0.05) lower TAC (on day −7, 0, +7 & +21), higher MDA (on day −21, −7, +7 and +35) and NO (on day +7, +21 & +35) concentrations compared to normal cows. The expression of CAT and GPx4 genes was lower (P<0.05) and SOD2 gene was higher (P<0.05) in endometrial tissue of cows that developed uterine infection compared to normal cows. The relationship of peripheral levels of MDA and NO with antioxidant enzymes expression in endometrial tissue was found significant. Receiver operator characteristic analysis revealed that the concentrations of TAC on day −7 to day +35, MDA on day −21 to day +7 and NO on the day of calving to day +35 were highly correlated to the development of postpartum uterine infection in cows. It may be inferred that the low serum TAC level and high level of lipid peroxidation and NO during peripartum period influenced the endometrial expression of anitioxidative genes that compromised the uterine health during postpartum period.

Increased mRNA expression of selected antimicrobial peptides around ovulation and during inflammatory processes in the bovine endometrium postpartum

In the uterus, the first pathogen confrontations take place at the luminal endometrial epithelium. Therefore, it is required that these cells have the potential to recognize and respond to a bacterial infection. Antimicrobial peptides (AMP), part of the innate immune system in addition to cytokines, are principal effector molecules of mucosal immunity against pathogens. One important family of AMP that can permeabilize bacterial membranes is the beta-defensin (DEFB) family, which includes the following members: DEFB1, DEFB4A, and DEFB5, lingual AMP, and tracheal AMP. The bactericidal/permeability-increasing protein is also a cationic AMP that results in the death of bacteria. Another AMP family is the S100 calcium-binding protein (S100A) family including the following members: S100A8, S100A9, S100A11, and S100A12. These AMP exert their antimicrobial action through chelation of several ions. The aim of the present study was to evaluate mRNA expression patterns of selected AMP in bovine endometrial cells collected (1) at different stages of the estrous cycle (postovulatory, early-to-mid luteal, late luteal, and pre-ovulatory phase); (2) during the puerperium depending on uterine health status (healthy, subclinical, or clinical endometritis) starting on Day 24 to 30 postpartum for 3 weeks on a weekly basis; and (3) in vitro after co-culturing with Bacillus pumilus at three different multiplicities of infection (MOI 1, 5, and 10) up to 6 hours. The results reported that the mRNA expression of all candidate AMP, except DEFB1, S100A8, and S100A9, was estrous cycle dependent. In particular, around the time of ovulation, the transcription level of most AMP was higher (P < 0.05) compared with the luteal phase. Almost all candidate AMP mRNA expression was dependent on uterine health status, with a higher transcription level (P < 0.05) in inflamed endometrial tissues, especially during the late stage of the puerperium (Day 45–51 postpartum). Members of the DEFB family were nearly unaffected in their mRNA expression in primary endometrial cells co-incubated with B. pumilus. However, S100A8 and S100A9 mRNA contents were higher after 4 and 6 hours of co-incubation with B. pumilus compared with untreated controls. In conclusion, higher mRNA expression of the candidate AMP around ovulation or in inflamed endometrial tissue during the puerperium suggests their crucial role in uterine innate immunity in the defense against invading bacteria.