Investigation of Uterine Fluid Extracellular Vesicles' Proteomic Profiles Provides Novel Diagnostic Biomarkers of Bovine Endometritis.

Cow uterine infections pose a challenge in dairy farming, resulting in reproductive disorders. Uterine fluid extracellular vesicles (UF-EVs) play a key role in cell-to-cell communication in the uterus, potentially holding the signs of aetiology for endometritis. We used mass spectrometry-based quantitative shotgun proteomics to compare UF-EV proteomic profiles in healthy cows (H), cows with subclinical (SE) or clinical endometritis (CLE) sampled at 28-35 days postpartum. Functional analysis was performed on embryo cultures with the exposure to different EV types. A total of 248 UF-EV proteins exhibited differential enrichment between the groups. Interestingly, in SE, EV protein signature suggests a slight suppression of inflammatory response compared to CLE-UF-EVs, clustering closer with healthy cows' profile. Furthermore, CLE-UF-EVs proteomic profile highlighted pathways associated with cell apoptosis and active inflammation aimed at pathogen elimination. In SE-UF-EVs, the regulation of normal physiological status was aberrant, showing cell damage and endometrial repair at the same time. Serine peptidase HtrA1 (HTRA1) emerged as a potential biomarker for SE. Supplementation of CLE- and SE-derived UF-EVs reduced the embryo developmental rates and quality. Therefore, further research is warranted to elucidate the precise aetiology of SE in cattle, and HTRA1 should be further explored as a potential diagnostic biomarker.

PI3K/AKT signaling alters glucose metabolism in uterine microenvironment of women with idiopathic recurrent spontaneous miscarriage.

BACKGROUND: This study aims to identify metabolomic signatures in uterine fluid of women with idiopathic recurrent spontaneous miscarriage (IRSM) during window of implantation (WOI). Also, glucose transporters GLUT3 and GLUT4 and proteins of PI3K-Akt signaling pathway in endometrial tissue are assessed. METHODS: Paired uterine fluid and endometrial biopsies were collected during WOI from women with IRSM (n = 24) and healthy women with azoospermic male partners as controls (n = 15). NMR metabolomics was used to identify the dysregulated metabolites in uterine fluid of IRSM women. Additionally, proteins and glucose transporters were investigated in the endometrial tissue using immunohistochemistry (IHC) and western blotting. RESULTS: Uterine fluid metabolomics indicated eleven metabolites to be significantly downregulated in IRSM. While expression levels of PI3K (p85), PI3K (p110), p-Akt (Thr308), p-Akt (Ser473), GLUT3 and GLUT4 were significantly downregulated in endometrial tissue of these women, p-IKK α/ß (Ser176/180) and p-NFkBp65 (Ser536) were significantly increased. CONCLUSION: Our findings suggest that dysregulation of PI3K/Akt pathway in the uterine microenvironment could be a likely cause of endometrial dysfunction, thereby affecting implantation. Further studies on the downstream effects of the Akt signaling pathway in-vitro for improved understanding of the Akt-mediated cellular responses in IRSM is, therefore, warranted.

PPARG is dispensable for bovine embryo development up to tubular stages.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors (PPARs) mediate the signaling actions of prostaglandins and other lipids and, between them, PPARG has been pointed out to play a relevant role on conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG KO embryos in vitro by two independent gene ablation strategies and assess their developmental ability. In vitro development to Day (D) 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass and trophectoderm cell numbers were similar between WT and KO D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, and embryonic disc formation and hypoblast migration rates were unaffected by the ablation. The development to tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disc was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

Assessment of immune cells in the uterine fluid at the time of the embryo transfer.

PROBLEM: Although endometrial receptivity is a key factor in influencing implantation in both naturally conceived and assisted reproductive technology (ART) cycles, very little is known about the endometrium milieu around the time of implantation. Previous studies have demonstrated the presence of several cytokines in the endometrium that affect implantation. However, there is lacking data about the presence of immune cell subtypes within the endometrium and in the uterine cavity at the time of implantation. METHOD OF STUDY: This study was approved by the Institutional Review Board (# 225589). The study was designed as a prospective observational cohort study between May 2021 and December 2022 at a single academic-based fertility center. All patients underwent at least one In Vitro Fertilization (IVF) cycle and have frozen embryos. Twenty-four participants were recruited for this study which was conducted during the frozen embryo transfer (FET) cycle regardless of the outcome of previous cycles. Two samples were acquired from each subject, denoted as lower and upper. A trial transfer catheter was introduced under ultrasound guidance into the lower uterine segment. Upon removal, the tip was rinsed in IMDM medium containing 10% FBS (lower uterus). A transfer catheter was then loaded with the embryo that was placed in the upper uterus under ultrasound guidance. The tip of the transfer catheter was rinsed in separate aliquot of the above media (upper uterus). After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD66b, CD14, CD16, CD56 and acquired on Sony SP6800 Spectral Analyzer. RESULTS: Upon staining the pelleted cells, we were able to identify viable leukocytes from samples obtained from both, upper and lower uterus (0.125 × 106 cells ± SD 0.32), (0.123 × 106 cells ± SD 0.12), respectively. Among total viable cells, there was no significant difference in both percent and number of CD45+ cells between the upper and lower uterus (9.88% ± 6.98 SD, 13.67% ± 9.79 SD, p = .198) respectively. However, there was significantly higher expression of CD3+ (p = .006), CD19+ (p = .032) and CD14+ (p = .019) cells in samples collected from upper compared to lower uterus. Within all CD3+ cells, we found that gamma delta T cells (GDT) were the major population of T cells in both upper and lower uterus. In contrast, CD8+ T cells were significantly higher in the lower uterus when compared to the upper uterus (p = .009). There was no statistically significant difference in the expression of CD4+ T cells, T regulatory cells (CD4+CD25+CD127-), NK cells (CD56+), neutrophils (CD66b+) and FcγRIII+ cells (CD16+) between upper and lower uterus. CONCLUSIONS: We believe the immune milieu at the time of embryo transfer will affect implantation. Understanding the composition of immune cells will guide further research in identifying optimal immune milieus that favor implantation. Comprehensive analysis of endometrium is expected to lead to new diagnostic and therapeutic approaches to improve IVF outcomes.

Revealing the mechanism of fiber promoting sow embryo implantation by altering the abundance of uterine fluid proteins: A proteomic perspective.

Many studies have shown that fiber in the diet plays an important role in improving the reproductive performance of sows, but there is rarely research on the impact of fiber on early embryo implantation. This study used 4D-Label free technology to identify and analyze the effect of the fiber composition in the diet on the protein in the early pregnancy uterine fluid (UF) of sows. The results indicate that ratio of insoluble fibers to soluble fibers (ISF/SF) 4.89 can increase the concentration of progesterone (PROG) and reduce tumor necrosis factorα (TNF-α) concentration in sow UF. In addition, through 4D-Label free, we identified a total of 4248 proteins, 38 proteins abundance upregulated and 283 proteins abundance downregulated in UF. Through enrichment analysis of these differential abundance proteins (DAPs), it was found that these differential proteins are mainly related to the docking of extracellular vesicles, vesicular transport, inflammatory response, and insulin resistance. Therefore, the results of this study reveal the possible mechanism by which fiber improves the reproductive performance of sows, laying a theoretical foundation for future research on the effects of diet on reproduction. SIGNIFICANCE: This study demonstrates the importance of dietary fiber for early embryo implantation in sows. The effect of dietary ISF/SF on early embryo implantation in sows was elucidated from a proteomic perspective through 4D-Label free technology. This study not only has significant implications for improving sow reproductive efficiency, but also provides important theoretical references for studying early miscarriage and reproductive nutrition in human pregnancy.

Ovarian Stimulation Altered Uterine Fluid Extracellular Vesicles miRNA Affecting Implantation in Rats.

Uterine fluid (UF) extracellular vesicle (EV) miRNA may affect implantation and could be the potential biomarker of endometrial receptivity (ER). Ovarian stimulation (OS) could damage the ER but its mechanism is still unclear. Here, we evaluate the affections of OS on UF EV miRNA expression and implantation. Female rats were divided into three groups: natural cycle or injection with GnRH-a following HP-HMG or u-FSH. UF was collected on the 5th day of gestation. Affinity membrane columns were utilized to isolate EVs from UF, obtained during implantation flushing. The EV miRNAs were sequenced, and five of them were validated by qRT-PCR. HTR-8/Svneo cells were transfected with miR-223-3p mimic and inhibitor, followed by conducting colony formation, invasion, migration, and adhesion assays to assess the cellular functions. In OS groups, the implantation rate decreased (p < 0.05), and the pinopode was damaged in the OS groups. The EVs were isolated from UF, and the differential expression key miRNAs were involved in several regulation pathways, such as cancer, endocrine, and cell cycles, which were correlated with ER and implantation. Among the miRNAs, miR-223-5p greatly differed and was most consistent with the sequencing results, followed by miR-223-3p and miR-98-5P. miR-223-3p promoted HTR-8/SVneo cells grow and ability of invasion, migration, and adhesion. OS altered UF EVs miRNAs affecting implantation in rats, and miR-223-3p might be the key molecule.

Uterine secretome: What do the proteins say about maternal-fetal communication in buffaloes?

The aim was to compare the UF proteomics of pregnant and non-pregnant buffalo during early pregnancy. Forty-four females were submitted to hormonal estrus synchronization and randomly divided into two groups: pregnant (n = 30) and non-pregnant (n = 14). The pregnant group was artificially inseminated and divided into a further two groups: P12 (n = 15) and P18 (n = 15). Conceptus and uterine fluid samples were collected during slaughter at, respectively, 12 and 18 days after insemination. Of all the inseminated females, only eight animals in each group were pregnant, which reduced the sample of the groups to P12 (n = 8) and P18 (n = 8). The non-pregnant group was also re-divided into two groups at the end of synchronization: NP12 (n = 7) and NP18 (n = 7). The UF samples were processed for proteomic analysis. The results were submitted to multivariate and univariate analysis. A total of 1068 proteins were found in the uterine fluid in both groups. Our results describe proteins involved in the conceptus elongation and maternal recognition of pregnancy, and their action was associated with cell growth, endometrial remodeling, and modulation of immune and antioxidant protection, mechanisms necessary for embryonic maintenance in the uterine environment. SIGNIFICANCE: Uterine fluid is a substance synthesized and secreted by the endometrium that plays essential roles during pregnancy in ruminants, contributing significantly to embryonic development. Understanding the functions that the proteins present in the UF perform during early pregnancy, a period marked by embryonic implantation, and maternal recognition of pregnancy is of fundamental importance to understanding the mechanisms necessary for the maintenance of pregnancy. The present study characterized and compared the UF proteome at the beginning of pregnancy in pregnant and non-pregnant buffaloes to correlate the functions of the proteins and the stage of development of the conceptus and unravel their processes in maternal recognition of pregnancy. The proteins found were involved in cell growth and endometrial remodeling, in addition to acting in the immunological protection of the conceptus and performing antioxidant actions necessary for establishing a pregnancy.

Different seminal ejaculated fractions in artificial insemination condition the protein cargo of oviductal and uterine extracellular vesicles in pig.

The seminal plasma (SP) is the liquid component of semen that facilitates sperm transport through the female genital tract. SP modulates the activity of the ovary, oviductal environment and uterine function during the periovulatory and early pregnancy period. Extracellular vesicles (EVs) secreted in the oviduct (oEVs) and uterus (uEVs) have been shown to influence the expression of endometrial genes that regulate fertilization and early embryo development. In some species, semen is composed of well-separated fractions that vary in concentration of spermatozoa and SP composition and volume. This study aimed to investigate the impact of different accumulative fractions of the porcine ejaculate (F1, composed of the sperm-rich fraction, SRF; F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF) on oEVs and uEVs protein cargo. Six days after the onset of estrus, we determined the oEVs and uEVs size and protein concentration in pregnant sows by artificial insemination (AI-sows) and in non-inseminated sows as control (C-sows). We also identified the main proteins in oEVs and uEVs, in AI-F1, AI-F2, AI-F3, and C-sows. Our results indicated that although the size of EVs is similar between AI- and C-sows, the protein concentration of both oEVs and uEVs was significantly lower in AI-sows (p < 0.05). Proteomic analysis identified 38 unique proteins in oEVs from AI-sows, mainly involved in protein stabilization, glycolytic and carbohydrate processes. The uEVs from AI-sows showed the presence of 43 unique proteins, including already-known fertility-related proteins (EZR, HSPAA901, PDS). We also demonstrated that the protein composition of oEVs and uEVs differed depending on the seminal fraction(s) inseminated (F1, F2, or F3). In conclusion, we found specific protein cargo in oEVs and uEVs according to the type of semen fraction the sow was inseminated with and whose functions these specific EVs proteins are closely associated with reproductive processes.

Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle.

Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics-based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle-dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle-dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development, and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion, and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased in vitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (p ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production in vitro.

Profiling the metabolome of uterine fluid for early detection of ovarian cancer.

Ovarian cancer (OC) causes high mortality in women because of ineffective biomarkers for early diagnosis. Here, we perform metabolomics analysis on an initial training set of uterine fluid from 96 gynecological patients. A seven-metabolite-marker panel consisting of vanillylmandelic acid, norepinephrine, phenylalanine, beta-alanine, tyrosine, 12-S-hydroxy-5,8,10-heptadecatrienoic acid, and crithmumdiol is established for detecting early-stage OC. The panel is further validated in an independent sample set from 123 patients, discriminating early OC from controls with an area under the curve (AUC) of 0.957 (95% confidence interval [CI], 0.894-1). Interestingly, we find elevated norepinephrine and decreased vanillylmandelic acid in most OC cells, resulting from excess 4-hydroxyestradiol that antagonizes the catabolism of norepinephrine by catechol-O-methyltransferase. Moreover, exposure to 4-hydroxyestradiol induces cellular DNA damage and genomic instability that could lead to tumorigenesis. Thus, this study not only reveals metabolic features in uterine fluid of gynecological patients but also establishes a noninvasive approach for the early diagnosis of OC.

Amniotic Mesenchymal-Derived Extracellular Vesicles and Their Role in the Prevention of Persistent Post-Breeding Induced Endometritis.

Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose-response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 106 EVs with 10 × 106 spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

Adverse effect of assisted reproductive technology-related hyperoestrogensim on the secretion and absorption of uterine fluid in superovulating mice during the peri-implantation period.

Objectives: This study aimed to investigate the potential mechanism of hyperoestrogensim elicited by ovulation induction affects endometrial receptivity and leads to embryo implantation abnormality or failure. Study design: Establishment of ovulation induction mouse model. Changes in mouse body weight, ovarian weight, serum E2 level and oestrous cycle were observed. During the peri-implantation period, morphological changes in the mouse uterus and implantation sites and the localization and protein levels of oestrogen receptors ERα and ERß, the tight junction factors CLDN3 and OCLN, the aquaporins AQP3, AQP4 and AQP8, and the sodium channel proteins SCNN1α, SCNN1ß and SCNN1γ were observed. The expression and cellular localization of ERα, CLDN3, AQP8 and SCNN1 ß in RL95-2 cell line were also detected by western blotting and immunofluorescence. Results: Ovarian and body weights were significantly higher in the 5 IU and 10 IU groups than in the CON group. The E2 level was significantly higher in the 10 IU group than in the CON group. The mice in the 10 IU group had a disordered oestrous cycle and were in oestrus for a long time. At 5.5 dpc, significantly fewer implantation sites were observed in the 10 IU group than in the CON (p<0.001) and 5 IU (p<0.05) groups. The probability of abnormal implantation and abortion was higher in the 10 IU group than in the CON and 5 IU groups. CLDN3, OCLN, AQP8 and SCNN1ß in the mouse endometrium were localized on the luminal epithelium and glandular epithelium and expression levels were lower in the 10 IU group than in the CON group. The protein expression level of ERα was increased by 50% in the 10 IU group compared to the CON group. The expressions of CLDN3, AQP8, SCNN1ß in RL95-2 cell line were significantly depressed by the superphysiological E2, ERα agonist or ERß agonist, which could be reversed by the oestrogen receptor antagonist. Conclusion: ART-induced hyperoestrogenism reduces CLDN3, AQP8 and SCNN1ß expression through ERα, thereby destroying tight junctions and water and sodium channels in the endometrial cavity epithelium, which may cause abnormal implantation due to abnormal uterine fluid secretion and absorption.

Effects of RFRP­3 on an ovariectomized estrogen­primed rat model and HEC­1A human endometrial carcinoma cells.

The hypothalamic peptide gonadotropin inhibitory hormone (GnIH) is a relatively novel hypothalamic neuropeptide, identified in 2000. It can influence the hypothalamic-pituitary-gonadal axis and reproductive function through various neuroendocrine systems. The present study aimed to explore the effects and potential underlying molecular mechanism of RFamide-related peptide-3 (RFRP-3) injection on the uterine fluid protein profile of ovariectomized estrogen-primed (OEP) rats using proteomics. In addition, the possible effects of RFRP-3 on the viability and apoptosis of the human endometrial cancer cell line HEC-1A and associated molecular mechanism were investigated. The OEP rat model was established through injection with GnIH/RFRP-3 through the lateral ventricle. At 6 h after injection, the protein components of uterine fluid of rats in the experimental and control groups were analyzed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Differentially expressed proteins (DEPs) were analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interactions (PPI) were investigated using the STRING database. PPI networks were then established before hub proteins were selected using OmicsBean software. The expression of one of the hub proteins, Kras, was then detected using western blot analysis. Cell Counting Kit-8, Annexin V-FITC/PI, reverse transcription-quantitative PCR and western blotting were also performed to analyze cell viability and apoptosis. In total, 417 DEPs were obtained using LC-MS/MS, including 279 upregulated and 138 downregulated proteins. GO analysis revealed that the majority of the DEPs were secretory proteins. According to KEGG enrichment analysis, the DEPs found were generally involved in tumor-associated pathways. In particular, five hub proteins, namely G protein subunit α (Gna)13, Gnaq, Gnai3, Kras and MMP9, were obtained following PPI network analysis. Western blot analysis showed that expression of the hub protein Kras was downregulated following treatment with 10,000 ng/ml RFRP-3. RFRP-3 treatment (10,000 ng/ml) also suppressed HEC-1A cell viability, induced apoptosis, downregulated Bcl-2 and upregulated Bax protein expression, compared with those in the control group. In addition, compared with those in the control group, RFRP-3 significantly reduced the mRNA expression levels of PI3K, AKT and mTOR, while upregulating those of LC3-II. Compared with those in the control group, RFRP-3 significantly decreased the protein expression levels of PI3K, AKT, mTOR and p62, in addition to decreasing AKT phosphorylation. By contrast, RFRP-3 significantly increased the LC3-II/I ratio and G protein-coupled receptor 147 (GPR147) protein expression. In conclusion, the present data suggest that RFRP-3 can alter the protein expression profile of the uterine fluid of OEP rats by upregulating MMP9 expression whilst downregulating that of key hub proteins Gna13, GnaQ, Gnai3 and Kras. Furthermore, RFRP-3 can inhibit HEC-1A cell viability while promoting apoptosis. The underlying molecular mechanism may involve activation of GPR147 receptor by the direct binding of RFRP-3, which further downregulates the hub protein Kras to switch on the PI3K/AKT/mTOR pathway. This subsequently reduces the Bcl-2 expression and promotes Bax expression to induce autophagy.

The bovine uterine fluid proteome is more impacted by the stage of the estrous cycle than the proximity of the ovulating ovary in the periconception period.

Uterine secretions provide a suitable environment for sperm selective migration during a couple of days preceding ovulation and for early embryo development before implantation. Our goal was to identify and quantify proteins in the bovine uterine fluid during the periovulatory period of the estrous cycle. Genital tracts with normal morphology were collected from adult cyclic Bos taurus females in a local slaughterhouse and classified into pre-ovulatory or post-ovulatory stages of cycle (around days 19-21 and 0-5 of cycle, respectively; n = 8 cows per stage) based on ovarian morphology. Proteins from uterine fluid collected from the utero-tubal junction to the base of each horn (four pools of two cows per condition) were analyzed by nanoLiquid Chromatography coupled with tandem Mass Spectrometry (nanoLC-MS/MS). A total of 1214 proteins were identified, of which 91% were shared between all conditions. Overall, 57% of proteins were predicted to be secreted and 17% were previously reported in uterine extracellular vesicles. Paired comparisons between uterine horns ipsilateral and contralateral to ovulation evidenced 12 differentially abundant proteins, including five at pre-ovulatory stage. Furthermore, 35 proteins differed in abundance between pre- and post-ovulatory stages, including 21 in the ipsilateral side of ovulation. Functional analysis of identified proteins demonstrated roles in binding, metabolism, cellular detoxification and the immune response. This study provides a valuable database of uterine proteins for functional studies on sperm physiology and early embryo development.

Serological and Uterine Biomarkers for Detecting Endometritis in Mares.

Serological analysis may provide relevant information on endometritis diagnostics. Therefore, mares scheduled for AI with refrigerated semen, at the time of heat signs, underwent blood and uterine fluid samplings using a swab, uterine lavage for culture analysis, and treatment with human chorionic gonadotropin to induce ovulation. After 24-28 h, the mares were inseminated and, if positive at the culture test, treated with antibiotics chosen based on the susceptibility test. Uterine cells obtained by swabs were used for cytological examination with both classical and fluorescence techniques. Blood serum and uterine fluid samples were analyzed for assessing parameters related to redox balance, inflammation, and protease regulator potential. In blood serum, total antioxidant capacity, measured as the ferric reducing ability of plasma (FRAP), was significantly lower in cytologically endometritis-positive than -negative mares. In the uterine fluid, total thiol levels (TTL), nitric oxide metabolites (NOx), protease activity and total protein content varied significantly between groups. Although the cytological examination was more capable of discriminating between endometritis-positive and -negative mares in relation to the parameters examined, no statistically significant differences emerged in terms of pregnancy rate in relation to cytological and culture diagnosis as well as in mares diagnosed as positive and negative for endometritis.

Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†.

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.

Characterization of bovine uterine fluid extracellular vesicles proteomic profiles at follicular and luteal phases of the oestrous cycle.

Extracellular vesicles (EV) have been identified in uterine fluid (UF), however the bovine UF-EV profile during different phases of the oestrous cycle has not yet been established. Therefore, we compared the UF-EV, and their protein profile at follicular and luteal phases of the oestrous cycle. UF samples were collected from healthy uteri of six live and six slaughtered cows at follicular or luteal phases. Isolation of EV was performed using tangential flow filtration followed by size exclusion chromatography. EV were characterized by nanoparticle tracking analysis (NTA), fluorescence NTA, zeta potential, and transmission electron microscopy. Mass-spectrometry was used to evaluate EV protein profile from live cows. Particle concentrations (mean ± SD) were higher (P < 0.05) at follicular than at luteal phase in both live (1.01 × 108 ± 1.66 × 107 vs 7.56 × 107 ± 1.80 × 107, respectively) and slaughtered cows (1.17 × 108 ± 2.34 × 107 vs 9.12 × 107 ± 9.77 × 106, respectively). The proportion of fluorescently labelled EV varied significantly between follicular and luteal phases across live (28.9 ± 1.9% vs 19.3 ± 2.8%, respectively) and slaughtered cows (26.5 ± 6.3% vs 27.3 ± 2 .7%, respectively). In total, 41 EV proteins were differentially expressed between the phases. Some of the proteins were involved in reproductive processes, cell adhesion and proliferation, and cellular metabolic processes. The results indicated differences in bovine UF-EV concentration and protein profile at follicular and luteal phases, which would suggest that EV modulate uterine microenvironment across the oestrous cycle. Further research is needed to understand the effect of EV changes throughout the oestrous cycle.

Effects of feeding strategies on eggshell quality of laying hens during late laying period.

Insufficient calcium supply during the dark period is an important reason for deteriorated eggshell quality in laying hens. In the present study, the feeding time of hens was altered in order to investigate whether the changes in feeding time and feed consumption could influence the laying performance and eggshell quality of hens. A total of 192, 60-wk-old Hy-line Brown hens with similar body weight and laying rate were obtained. The hens were randomly divided into 4 groups and subjected to the following feeding strategies: feeding 3 times a day (control group, CON), or feeding once a day in the morning at 08:00 (MF), in the noon at 12:00 (NF), or in the afternoon at 16:00 (AF), respectively. The feeding strategies had no significant effect (P > 0.05) on laying rate, egg weight, and egg mass. Although the feed intake did not differ among treatments, the time phase of feed consumption was changed. From 15:00 to 21:00 h, hens consumed 49.7%, 42.4%, 49.1%, and 70.8% of daily feed intake in the CON, MF, NF, and AF groups, respectively. Feeding strategy had no detectable influence (P > 0.05) on egg shape index, eggshell strength, and eggshell percentage. Compared to CON, AF hens tended to have a higher eggshell thickness (P = 0.053). In MF and NF treatments, plasma calcium (Ca), phosphorus (P) levels, and alkaline phosphatase (ALP) activity did not differ (P > 0.05) compared with CON. In contrast, AF-hens had lower Ca and P levels, but a higher ALP activity than CON (P < 0.01). The AF hens had higher uterine fluid Ca than MF and NF hens (P < 0.05). Compared to CON, the expression level of CaBP-D28K was increased in the shell gland mucosa of MF-hens. Also, MF-, NF-, and AF-hens had higher Osteopontin (OPN) expression level (P < 0.05), whereas NF had a higher expression of OC-116 (P < 0.01). In conclusion, the results indicated that feeding in the afternoon changed the pattern of feed consumption and exerted a positive influence on eggshell thickness.

Safety and feasibility of PVA formaldehyde absorbent sponge in noninvasive uterine fluid sampling and RNA sequencing. / PVAç¼©ç”²é†›å¸æ°´æµ·ç»µåœ¨æ— åˆ›å®«è ”æ¶²å–æ ·å’ŒRNAæ£€æµ‹ä¸­çš„å®‰å ¨æ€§ä¸Žå¯è¡Œæ€§.

OBJECTIVES: Uterine fluid RNA can be used as a test for endometrial receptivity, but there is still no noninvasive sampling method available. The polyvinyl alcohol (PVA) formaldehyde absorbent sponge, a medical bio-absorbent sponge with good water absorption and biophilic properties, can be used to develop a new noninvasive endometrial fluid sampler. This study aims to investigate the toxicity of PVA acetal absorbent sponges on endometrial epithelial cells and its effect on RNA sequencing (RNA-Seq). METHODS: The experimental group using PVA formaldehyde absorbent sponge was prepared into 0.005%, 0.01% and 0.02% (w/v) suspension, and 0.01%, 0.05% and 0.1% (v/v) extract groups. The control group was only the complete culture medium. Nothing was added to the blank group. In vitro cytotoxicity assay was used to evaluate the survival rate of cells. Eight patients underwent in vitro fertilization treatment in the Reproductive Center of Xiangya Hospital, Central South University from November 2019 to January 2020. The uterine fluid of each patient was aspirated. The experimental group was inhaled with sterile PVA formaldehyde absorbent sponge and then immersed RNA-later solution. The control group was directly injected into the same amount of RNA-later solution. RNA-seq and data analysis was performed later. RESULTS: The vitro cytotoxicity assay showed that in suspension groups, there was no significance difference in cell survival between different co-culture time in 0.005% group (P=0.255). In the 0.01% and 0.02% group, there was no difference at each incubation time within 12 h (all P>0.05), but the cell survival rate was decreased at 24 h compared with 0 h (P<0.01, P<0.05). At the same co-culture time, the cell survival of the 3 concentration gradient groups were significantly lower than that of the control group (all P<0.05). The cell viability of the 0.005% concentration group was decreased less than 30% at 24 h, the 0.01% concentration group decreased more than 30% at 12 h, and the 0.02% concentration group was decreased more than 30% at 0 h. For extract groups, there was no significant difference in the survival rate within 6 h in 0.01% concentration group (all P>0.05), and the survival rate of 12 h and 24 h was lower than that of 0 h group (both P<0.01). In 0.05% group, there was no significant difference at each incubation time within 12 h (all P>0.05), but the survival rate at 24 h was lower than that at 0 h (P<0.05). There was no significant difference in survival rate at different culture time in 0.1% concentration group (P=0.082). At the same culture time, there was no significant difference in survival rate between 0.01% group and control group at 0, 3 and 24 h (all P>0.05). Except for 3 h, the survival rate of 0.05% and 0.1% groups was lower than that of control group (all P<0.05), and the decrease was all less than 30%. Uterine fluid RNA-seq showed that there was no significance difference in exonic rate, the detected genes and transcripts of RNA between the experiment groups and the control group (all P>0.05). CONCLUSIONS: The in vitro cytotoxic of PVA formaldehyde absorbent sponge on human endometrial epithelial cell meet the national standard of the cytotoxic of medical materials. Sampling the uterine fluid with this material does not affect the RNA-Seq results. PVA formaldehyde absorbent sponge is safe and feasible when appling to the noninvasive uterine fluid sampling and RNA sequencing.

Tumor Necrosis Factor Alpha Level in the Uterine Fluid of Patients with Polycystic Ovary Syndrome and Its Correlation with Clinical Parameters.

Objective: This study aimed to analyze tumor necrosis factor alpha (TNF-α) level in the uterine fluid of patients with polycystic ovary syndrome (PCOS) and its correlation with the clinical parameters of PCOS. Methods: A total of 162 patients treated in the Reproductive Medicine Center of the General Hospital of Ningxia Medical University between December 2019 and November 2021 were enrolled as research subjects, including 80 patients with PCOS and 82 patients with other gynecological disease, who were used as the controls. The patients' general data, along with blood glucose, blood lipid, insulin, and sex hormone levels and other data, were collected. The TNF-α levels in the patients' serum and uterine fluid were detected using enzyme-linked immunosorbent assay. Results: Compared with the patients in the control group, the body mass index (BMI), anti-Müllerian hormone, luteinizing hormone, testosterone (T), fasting insulin (FINS), homeostasis model assessment insulin resistance (HOMA-IR), triglyceride (TG), and low-density lipoprotein (LDL) of patients with PCOS were higher, and high-density lipoprotein was lower (P < 0.05). The TNF-α levels in the serum and uterine fluid of patients with PCOS were higher than those in the control group (P < 0.01), and the TNF-α levels in the uterine fluid of these patients was significantly correlated with BMI, T, FINS, HOMA-IR, serum TNF-α, TG, and LDL (P < 0.05). Conclusion: There is local inflammation in the uterine cavity of patients with PCOS, and the detection of cytokines in uterine secretions may be a simple and feasible method of understanding the uterine microenvironment of patients with PCOS.