Endometrial Epithelium Research Articles

Comparative study on lesions of reproductive disorders of cows and female dromedary camels slaughtered at Addis Ababa, Adama and Akaki abattoirs with bacterial isolation and characterization

BackgroundReproduction is a basic prerequisite to efficient livestock production. Reproductive performance depends on the normal structure and function of genital organs. A cross-sectional study was conducted from November 2016 to May 2017 to identify and compare the frequency of reproductive tract pathological lesions and to isolate bacteria associated to uterine lesions in female dromedary camels and cows slaughtered at Akaki camel slaughterhouse and Addis Ababa and Adama municipal abattoirs. Purposive sampling technique was employed to include and examine the reproductive tracts of all slaughtered animals (280; 140 cows and 140 camels) during the study period.ResultThe study examined a total of 280 (140 cows and 140 camels) reproductive tracts. Various pathological lesions with different degrees of severity were observed in 48 (34.2%) and 51 (36.4%) of dromedary camels and cows, respectively. In dromedary camels, the most prevalent lesion was uterine lesions (21.4%) followed by ovarian lesions (7.14%); while in cows, ovarian lesions were the most prevalent (16.4%) followed by uterine lesions (14.2%). In general, 56 bacteria were isolated from cows’ uterine lesion, the Staphylococcus species (28.5%), Streptococci species (19.6%), Coynebacterium species (8.9%), Escherichia coli (26.78%), Salmonella species (10.7%) and Klebsiella species (5.35%) being the most representative isolates. In camels, however, 45 bacteria were isolated from uterine lesions with higher prevalence of Escherichia coli (35.5%), Staphylococcus species (26.6%), Streptococcus species (13.3%), Pseudomonas species (6.6%), Proteus species (4.4%), Salmonella species (8.8%) and Klebsiella species (4.4%). Bacteriological data showed that the major isolates were similar, although slightly more frequent in occurrence in cows. Microscopically, uterine inflammatory lesions evidenced endometrial glands degeneration, epithelium sloughing, peri-glandular cuffing, and inflammatory cells infiltration.ConclusionsIn female dromedary camels and cows, pathological lesions of the reproductive tract showed great prevalence, with similarity in bacterial isolates in both species. The role of each reproductive lesion and bacterial isolates as causal agents of reproductive failures in these livestock species, however, needs further investigation.

How Does Endometriosis Lead to Ovarian Cancer? The Molecular Mechanism of Endometriosis-Associated Ovarian Cancer Development.

Simple SummaryDriver gene mutations have been identified in not only various types of endometriosis which are considered the origin of endometriosis-associated ovarian cancer but also the normal endometrium which is considered the origin of endometriosis. We focused on genomic linkage from normal endometrium to ovarian endometriosis and endometriosis-associated ovarian cancer (EAOC) and summarized the current knowledge of the commonality and differentiation of genomic features in the uterine endometrium, endometriosis, and EAOC. In addition, we have proposed molecular mechanism of ovarian carcinogenesis from the normal endometrium via endometriosis based on genomic alterations. This review is expected to contribute to research for the prevention of endometriosis and EAOC.Numerous epidemiological and histopathological studies support the notion that clear cell and endometrioid carcinomas derive from ovarian endometriosis. Accordingly, these histologic types are referred to as “endometriosis-associated ovarian cancer” (EAOC). Although the uterine endometrium is also considered an origin of endometriosis, the molecular mechanism involved in transformation of the uterine endometrium to EAOC via ovarian endometriosis has not yet been clarified. Recent studies based on high-throughput sequencing technology have revealed that cancer-associated gene mutations frequently identified in EAOC may exist in the normal uterine endometrial epithelium and ovarian endometriotic epithelium. The continuum of genomic alterations from the uterine endometrium to endometriosis and EAOC has been described, though the significance of cancer-associated gene mutations in the uterine endometrium or endometriosis remains unclear. In this review, we summarize current knowledge regarding the molecular characteristics of the uterine endometrium, endometriosis, and EAOC and discuss the molecular mechanism of cancer development from the normal endometrium through endometriosis in an effort to prevent EAOC.

Differential expression of protein disulfide isomerase (PDI) in regulating endometrial receptivity in humans

Estrogen and progesterone regulate the expression of endometrial proteins that determine endometrial receptivity for embryo implantation. The protein disulfide isomerase (PDI) family of proteins play a diverse role in regulating protein modification and redox function. Although the role of PDIs in cancer progression has been widely studied, their role in endometrial receptivity is largely unknown. We have focused on the expressions of PDIA1, PDIA2, PDIA3, PDIA4, PDIA5, and PDIA6 isoforms in endometrial epithelium under the influence of estrogen and progesterone and investigated their functional role in regulating endometrial receptivity. We found PDIA1−6 transcripts were expressed in endometrial epithelial Ishikawa, RL95-2, AN3CA, and HEC1-B cell lines. The expression of PDIA1 was low and PDIA5 was high in HEC1-B cells, whereas PDIA2 was high in both AN3CA and HEC1-B cells. In Ishikawa cells, estrogen (10 and 100 nM) upregulated PDIA1 and PDIA6, whereas estrogen (100 nM) downregulated PDIA4 and PDIA5; and progesterone (0.1 and 1 μM) downregulated transcript expressions of PDIA1–6. In human endometrial samples, significantly lowered transcript expressions of PDIA2 and PDIA5 were observed in the secretory phase compared with the proliferative phase, whereas no change was observed in the other studied transcripts throughout the cycle. Inhibition of PDI by PDI antibody (5 and 10 μg/mL) and PDI inhibitor bacitracin (1 and 5 mM) significantly increased the attachment of Jeg-3 spheroids onto AN3CA cells. Taken together, our study suggests a role of PDI in regulating endometrial receptivity and the possibility of using PDI inhibitors to enhance endometrial receptivity.

Size-dependent biodistribution of thiol-organosilica nanoparticles and F4/80 protein expression in the genital tract of female mice after intravaginal administration.

Recently the vaginal route consider as an ideal route for drug delivery systems (DDS) administration. This is because, it is suitable for lower drug dosage, higher drug concentration in the genital tract tissues and lower drug concentration in pregnant women blood circulation. However, the vaginal route administration faces many challenges due to the physiology as well as the complexity of vaginal tissue histology. Here in this study, during diestrus stage (optimal condition for foreign substance internalization), single or dual size of fluorescent thiol-organosilica nanoparticles (tOS-NPs) were administrated intravaginally. The biodistribution and reactivity of tOS-NPs in different tissues of the female genital tract were investigated under the fluorescence microscope. Furthermore, using immunohistochemical staining, the expression of F4/80 protein and the role of macrophages in transport and re-location of tOS-NPs from vaginal lumen into different genital tissues or other organs were investigated. This study showed that, tOS-NPs size and type of tissue are important in biodistribution and uptake of tOS-NPs in the genital tract. Small size (100nm) of tOS-NPs was highly accumulated in the genital tract tissues especially endometrial epithelium compared with large tOS-NPs (1000nm). Contradictory, the large size induced the expression of F4/80 protein and the number of vaginal macrophages compared with small size. However, both small and large sizes of tOS-NPs were found co-localized with F4/80+ macrophages, located in the vaginal, endometrial and ovarian tissues. The tOS-NPs intravaginally administrated were found in the splenic tissues, indicating its ability to enter the blood circulation from the vaginal lumen. Additionally, the high accumulation of tOS-NPs in the endometrial epithelium indicated the endometrial first pass effect of tOS-NPs. As a result, high concentration of tOS-NPs in the endometrial epithelium may reduce the concentration of tOS-NPs-based DDS in the blood circulation and their side effects. Furthermore, during vaginal tissue optimal condition (diestrus stage), understanding the fate and biodistribution of tOS-NPs will introduce important data about the development of save and effective DDS for the pregnant women.

Par3/integrin β1 regulates embryo adhesion via changing endometrial luminal epithelium polarity†.

The objective is to investigate the pathophysiological significance of Par3 and integrin β1 with regard to the functionality of the endometrial luminal epithelium (LE). Design: laboratory study; setting: university research laboratory. Analysis involved endometrial aspirates and endometrial adenocarcinoma cells (HEC-1A) and endometrial carcinoma cells (RL95-2). We first examined the expression and localization of Par3 and integrin β1 in HEC-1A cells and RL95-2 cells. Then we knocked down Par3 and integrin β1 in HEC-1A cells and RL95-2 cells, respectively, and found that Par3/integrin β1 affected embryo adhesion by regulating the intercellular tight junctions' (TJs') structure and thus the polarity of the endometrial LE. These findings were also confirmed in the endometrium specimens from human and mice. The main outcome measures were the expression and localization of Par3 and integrin β1 in the endometrial epithelial cell lines and endometrium specimens and the regulations of Par3 and integrin β1 on TJs, polarity, and embryo adhesion. Following the knockdown of Par3 in HEC-1A cells, there was a reduction in the complexity of the TJs and cell polarity, and the adhered blastocysts number was significantly increased. However, the reduction of integrin β1 in RL95-2 cells resulted in effects that directly opposed those following the knockdown of Par3 in HEC-1A cells. Estrogen and progesterone reduced the expression of Par3 and promoted the expression of integrin β1 in HEC-1A cells. Par3/integrin β1 regulates embryo adhesion by regulating intercellular TJs' structure and polarity of endometrial LE under the action of ovarian hormones.

Label-free proteomics uncovers SMC1A expression is Down-regulated in AUB-E

BackgroundWhile heavy menstrual bleeding (HMB) is a prevalent symptom among women with abnormal uterine bleeding caused by endometrial disorder (AUB-E) seeking gynecologic care, the primary endometrial disorder remains poorly understood.MethodsFive human endometrial samples from women with AUB-E and the age-matched healthy women were selected, respectively. Proteins from the samples were analyzed by a linear ion trap (LTQ)-Orbitrap Elite mass spectrometer based label-free proteomic approach. The purpose protein was validated by western blot and immunohistochemistry staining.ResultsA total of 2353 protein groups were quantified under highly stringent criteria with a false discovery rate of < 1% for protein groups, and 291 differentially expressed proteins were significantly changed between the two groups. The results showed that the down-regulation of structural maintenance of chromosomes protein 1A (SMC1A) in AUB-E patients. Next, this change in the glandular epithelial cells was validated by immunohistochemistry.ConclusionThe results indicated a novel mechanism for the cause of AUB-E, as down-expression SMC1A potentially regulated the cell cycle progression in endometrial glandular epithelium further led to bleeding.

Histomorphological and Functional Features of the Eutopic Endometrium in Patients with Ovarian Endometriosis After Surgery-a Clinical Study.

Endometriosis causes infertility and the alterations in endometrial receptivity. Pinopodia in eutopic endometrial epithelium may have significant implications in the endometriosis-associated infertility. The aim of this study is to ascertain whether the surgical interventions to remove endometrioid ovarian cysts (EOCs) can improve endometrial receptivity. The study included 172 patients of reproductive age with EOC, who underwent laparoscopic cystectomy. Aspiration endometrial biopsy was performed at 6 and 12 months after the surgery during the proliferation and secretion phases. Histopathology analysis included H&E staining and IHC. Morphometric studies were performed on endometrial biopsies collected during the proliferation phase of 28 patients, and the secretion phase of 12 patients. The expression of IHC markers for estrogen receptors (ER) and progesterone receptors (PR) and the percentage of cells containing pinopodia were determined. A significant increase in the ER and PR expression was observed in the epithelium during the "middle stage, proliferation phase" and in the stroma and glands during "middle stage, secretion phase". A delay in endometrial secretory transformation and statistically significant decrease in the number of pinopodia was observed on the apical surface of the cells. These structural and functional alterations were observed both at 6 and 12 months after cystectomy. The endometriosis-associated infertility after surgical intervention of EOC could be due to the extensive expression of ER and PR during the proliferation and secretion phases, as well as the delayed secretory transformation and impaired formation of pinopodia in the eutopic endometrium in the patients at 6 and 12 months after surgery.

Endometrial regeneration with endometrial epithelium: homologous orchestration with endometrial stroma as a feeder

BackgroundThin endometrium adversely affects reproductive success rates with fertility treatment. Autologous transplantation of exogenously prepared endometrium can be a promising therapeutic option for thin endometrium; however, endometrial epithelial cells have limited expansion potential, which needs to be overcome in order to make regenerative medicine a therapeutic strategy for refractory thin endometrium. Here, we aimed to perform long-term culture of endometrial epithelial cells in vitro.MethodsWe prepared primary human endometrial epithelial cells and endometrial stromal cells and investigated whether endometrial stromal cells and human embryonic stem cell-derived feeder cells could support proliferation of endometrial epithelial cells. We also investigated whether three-dimensional culture can be achieved using thawed endometrial epithelial cells and endometrial stromal cells.ResultsCo-cultivation with the feeder cells dramatically increased the proliferation rate of the endometrial epithelial cells. We serially passaged the endometrial epithelial cells on mouse embryonic fibroblasts up to passage 6 for 4 months. Among the human-derived feeder cells, endometrial stromal cells exhibited the best feeder activity for proliferation of the endometrial epithelial cells. We continued to propagate the endometrial epithelial cells on endometrial stromal cells up to passage 5 for 81 days. Furthermore, endometrial epithelium and stroma, after the freeze-thaw procedure and sequential culture, were able to establish an endometrial three-dimensional model.ConclusionsWe herein established a model of in vitro cultured endometrium as a potential therapeutic option for refractory thin endometrium. The three-dimensional culture model with endometrial epithelial and stromal cell orchestration via cytokines, membrane-bound molecules, extracellular matrices, and gap junction will provide a new framework for exploring the mechanisms underlying the phenomenon of implantation. Additionally, modified embryo culture, so-called “in vitro implantation”, will be possible therapeutic approaches in fertility treatment.

Peptidoglycan Switches Off the TLR2-Mediated Sperm Recognition and Triggers Sperm Localization in the Bovine Endometrium.

In mammals, the uterine mucosal immune system simultaneously recognizes and reacts to most bacteria as well as allogenic sperm mainly through the Toll-like receptors (TLR)2/4 signaling pathway. Here, we characterized the impact of pathogen-derived TLR2/4 ligands (peptidoglycan (PGN)/lipopolysaccharide (LPS)) on the immune crosstalk of sperm with the bovine endometrial epithelium. The real-time PCR analysis showed that the presence of low levels of PGN, but not LPS, blocked the sperm-induced inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. Immunoblotting analysis revealed that PGN prevented the sperm-induced phosphorylation of JNK in BEECs. Activation or blockade of the TLR2 system in the endometrial epithelium verified that TLR2 signaling acts as a commonly-shared pathway for PGN and sperm recognition. The impairment of endometrial sperm recognition, induced by PGN, subsequently inhibited sperm phagocytosis by polymorphonuclear neutrophils (PMNs). Moreover, using an ex vivo endometrial explant that more closely resembles those in vivo conditions, showed that sperm provoked a mild and reversible endometrial tissue injury and triggered PMN recruitment into uterine glands, while PGN inhibited these events. Of note, PGN markedly increased the sperm attachment to uterine glands, and relatively so in the surface epithelium. However, addition of the anti-CD44 antibody into a PGN-sperm-explant co-culture completely blocked sperm attachment into glands and surface epithelia, indicating that the CD44 adhesion molecule is involved in the PGN-triggered sperm attachment to the endometrial epithelium. Together, these findings demonstrate that, the presence of PGN residues disrupts sperm immune recognition and prevents the physiological inflammation induced by sperm in the endometrial epithelium via the MyD88-dependent pathway of TLR2 signaling, possibly leading to impairment of uterine clearance and subsequent embryo receptivity.

Abstract PO048: Inhibition of P300 histone acetyltransferase activity rescues invasive phenotypes of ARID1A-deficient endometrial epithelium

Abstract Invasion is a hallmark feature of endometrial pathologies, including myometrial invasion of endometrial cancer and invasion of distal sites during endometriosis. Mutations in the chromatin remodeling protein ARID1A have been observed at high rates in endometrial cancer and endometriosis. ARID1A loss, in combination with phosphoinositide 3-kinase (PI3K) pathway activation, has been shown to induce invasion phenotypes in the endometrial epithelium. Using bioinformatic approaches, we identified histone acetyltransferase (HAT) protein P300 as potential co-regulator of ARID1A-deficient gene expression programs. Knockdown of P300 prevented invasive phenotypes in a genetically engineered mouse model and human endometrial epithelial cells through induction of apoptosis, indicating a synthetic-lethal relationship between ARID1A and P300 in the endometrial epithelium. Using the novel HAT inhibitor, A-485, we identified P300 HAT activity as the mechanism by which P300 promotes invasive phenotypes. A-485 treatment rescued invasion in vitro at concentrations which had no effect on cell viability in adherent culture, but induced anoikis in suspension culture. Using an RNA-seq approach, we identified cell adhesion and migration pathways as being regulated by ARID1A knockdown and P300 co-knockdown or A-485 co-treatment. Among genes in these pathways, we identified SERPINE1 as an essential component of P300-dependent invasive processes. SERPINE1 is overexpressed following ARID1A loss in mouse and human models, and SERPINE1 knockdown rescued invasive phenotypes following ARID1A loss. By chromatin immunoprecipitation, we identified a role for P300-dependent acetylation of histone 3 lysine 27 (H3K27ac) at the SERPINE1 super-enhancer. The novel P300 bromodomain inhibitor CCS1477 also rescues invasion and SERPINE1 expression following ARID1A loss, suggesting a role for bromodomain inhibition as a modulator of P300 H3K27ac deposition. These studies suggest the therapeutic utility of P300 inhibition to induce anoikis in mutant endometrium which has detached from the basement membrane, thereby preventing invasion. Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Inhibition of P300 histone acetyltransferase activity rescues invasive phenotypes of ARID1A-deficient endometrial epithelium [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO048.

Abstract PO008: ARID1A prevents H3K27-hyperacetylation at active super-enhancers

Abstract Mutations in the multi-subunit SWI/SNF (BAF) chromatin remodeling complex are observed in nearly all types of cancer. The most highly mutated subunit across cancer is ARID1A, which occurs in roughly half of uterine endometrial cancer cases. ARID1A mutations and loss of expression are also common in endometriosis. We have previously demonstrated that ARID1A loss drives transcriptional alterations leading to invasive phenotypes through multiple model systems. Yet, how ARID1A regulates transcription through chromatin regulation is poorly understood. We have generated genome-wide profiles of seven chromatin features associated with transcriptional regulation in human endometrial epithelial cells, in both the presence and absence of ARID1A, in order to identify a mechanism by which ARID1A regulates chromatin. Unbiased genome segmentation using these features revealed that ARID1A interacts with chromatin at highly active and accessible enhancers genome-wide. Out of all tested chromatin features, ARID1A most strongly co-localizes with H3K27ac, a mark of transcriptional activation at proximal and distal enhancers. ARID1A depletion also displayed the greatest effect on genomic H3K27ac as opposed to other histone modifications, H3K18ac, H3K4me1, H3K4me3, and H3K27me3. While the majority of typical enhancers had reduced H3K27ac with ARID1A loss, super-enhancers marked by high basal H3K27ac levels displayed hyperacetylation following ARID1A loss. Moreover, increasing H3K27ac was also associated with heightened eRNA transcription, indicating enhancer hyperactivation. Finally, RNA-seq revealed that genes associated with active super-enhancers were preferentially dysregulated following ARID1A loss. These results suggest that ARID1A-SWI/SNF differentially regulate typical enhancers vs. super-enhancers in the endometrial epithelium, and ARID1A may normally restrict super-enhancer hyperactivation to maintain epithelial identity and prevent endometrial invasion. Citation Format: Jake J. Reske, Mike R. Wilson, Ronald L. Chandler. ARID1A prevents H3K27-hyperacetylation at active super-enhancers [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO008.

Abstract IA007: The mutational landscape of normal human endometrial epithelium

Abstract Human endometrium is a highly dynamic tissue that undergoes numerous cycles of breakdown, rapid repair and remodelling in response to the oscillating levels of oestrogen and progesterone. The marked regenerative capacity of the tissue’s epithelial compartment is maintained by the intra-glandular adult stem cells (ASCs) that are retained during menstruation. Although the endometrial epithelial ASCs were first described over a decade ago, they remain poorly characterised in comparison to their counterparts in other tissues, such as the small and arge intestine. In particular, the size of the stem cell pool within individual glands, clonal dynamics and genomic landscape are largely unknown. In this study, we laser-capture microdissected 257 morphologically normal endometrial glands from pre- and post-menopausal women (aged 19 to 81). In selected donors, we also obtained samples of normal endocervical and Fallopian tubular epithelium. Isolated cellular material was subjected to our modified library preparation protocol and subsequent whole genome sequencing (WGS). Analysis of the WGS data identified that the majority of the glands were clonal cell populations sharing a recent common ancestor. Somatic mutations accumulated at a rate of 29 substitutions per year. Total mutation burden in the normal endometrial epithelium is higher than in other normal cells and manyfold lower than in endometrial cancers. Overall, despite the heterogeneity in age, reproductive history and BMI, we found relatively homogenous mutational processes to be operative in this tissue with only occasional outliers. Remarkably, we not only identify recurrent acquisitions of certain cancer-associated mutations, particularly those that are advantageous to cell growth, proliferation and migration, but also show that such events occur early in life, potentially even before adolescence. Using a generalised linear mixed effect model, we found that age has a positive association with the accumulation of driver mutations, while parity has a negative association. Over time, the mutant ASCs serve as a reservoir for the acquisition of further driver mutations to the extent that in some cases, the entire sampled endometrium becomes ‘neoplastic’ on the genomic level while still retaining the apparently normal phenotype. Our data identify a distinct mutational landscape in normal endometrium that is in keeping with the presence of early positive selection in this highly regenerative tissue. Citation Format: Luiza Moore. The mutational landscape of normal human endometrial epithelium [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA007.

Abstract IA020: Chromatin regulation of the endometrium: What are our models telling us?

Abstract ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. ARID1A is a SWI/SNF chromatin-remodeling complex subunit. ARID1A loss in the mouse endometrial epithelium leads vaginal bleeding when combined with PI3K pathway activation. Sorted mutant endometrial epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT), inflammation and estrogen signaling in vivo. ARID1A loss leads to the expression of EMT genes, suggesting ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates. In contrast to its repressive roles, ARID1A is required for the activation estrogen responsive genes, including the progesterone receptor (PGR). Estrogen responsive genes are marked by bivalent chromatin domains containing both histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3). High H3K27me3 correlates with low PGR expression, suggesting Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 normally silences PGR. In support of this, pharmacological inhibition of PRC2 partially rescues PGR activation is estrogen treated, ARID1A-deficient cells. Given the known antagonistic relationship between SWI/SNF and PRC2 complexes, we propose that SWI/SNF normally opposes PRC2 mediated silencing of the PGR promoter in response to estrogen, leading to PGR expression. In the ARID1A mutant endometrium, PGR promoter silencing by PRC2 is maintained. In the absence of PGR, progesterone cannot oppose the actions estrogen in the endometrium, leading to unopposed estrogen signaling. Our data demonstrate ARID1A has both activating and repressive roles in the endometrium, and ARID1A loss contributes to multiple aspects of disease pathogenesis. Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Chromatin regulation of the endometrium: What are our models telling us? [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA020.

Integrating LCM-Based Spatio-Temporal Transcriptomics Uncovers Conceptus and Endometrial Luminal Epithelium Communication that Coordinates the Conceptus Attachment in Pigs.

Attachment of conceptus to the endometrial luminal epithelium (LE) is a critical event for early placentation in Eutheria. Since the attachment occurs at a particular site within the uterus, a coordinated communication between three spatially distinct compartments (conceptus and endometrial LE from two anatomical regions of the uterus to which conceptus attaches and does not attach) is essential but remains to be fully characterized. Using the laser capture microdissection (LCM) technique, we firstly developed an approach that can allow us to pair the pig conceptus sample with its nearby endometrial epithelium sample without losing the native spatial information. Then, a comprehensive spatio-temporal transcriptomic profile without losing the original conceptus-endometrium coordinates was constructed. The analysis shows that an apparent difference in transcriptional responses to the conceptus exists between the endometrial LE from the two anatomically distinct regions in the uterus. In addition, we identified the communication pathways that link the conceptus and endometrial LE and found that these pathways have important roles in conceptus attachment. Furthermore, a number of genes whose expression is spatially restricted in the two different anatomical regions within the uterus were characterized for the first time and two of them (SULT2A1 and MEP1B) may cooperatively contribute to establish conceptus attachment in pigs. The results from our study have implications in understanding of conceptus/embryo attachment in pigs and other large polytocous species.

Regulation of ARHGAP19 in the endometrial epithelium: a possible role in the establishment of uterine receptivity

BackgroundThe establishment of uterine receptivity is essential for embryo implantation initiation and involves a significant morphological transformation in the endometrial epithelial cells (EECs). The remodeling of junctional complexes and membrane-associated cytoskeleton is crucial for epithelial transformation. However, little is known about how this process is regulated in EECs during the receptive phase. ARHGAP19 is a Rho GTPase-activating protein that participates in various cytoskeletal-related events, including epithelial morphogenesis. Here, we investigated the role of ARHGAP19 in endometrial epithelial transformation during the establishment of uterine receptivity. The upstream regulator of ARHGAP19 was also investigated.MethodsARHGAP19 expression was examined in mouse uteri during early pregnancy and in human EEC lines. The role of ARHGAP19 was investigated by manipulating its expression in EECs. The effect of ARHGAP19 on junctional proteins in EECs was examined by western blotting and immunofluorescence. The effect of ARHGAP19 on microvilli was examined by scanning electron microscopy. The upstream microRNA (miRNA) was predicted using online databases and validated by the dual-luciferase assay. The in vivo and in vitro effect of miRNA on endogenous ARHGAP19 was examined by uterine injection of miRNA agomirs and transfection of miRNA mimics or inhibitors.ResultsARHGAP19 was upregulated in the receptive mouse uteri and human EECs. Overexpression of ARHGAP19 in non-receptive EECs downregulated the expression of junctional proteins and resulted in their redistribution. Meanwhile, upregulating ARHGAP19 reorganized the cytoskeletal structure of EECs, leading to a decline of microvilli and changes in cell configuration. These changes weakened epithelial cell polarity and promoted the transition of non-receptive EECs to a receptive phenotype. Besides, miR-192-5p, a miRNA that plays a key role in maintaining epithelial properties, was validated as an upstream regulator of ARHGAP19.ConclusionThese results suggested that ARHGAP19 may contribute to the transition of EECs from a non-receptive to a receptive state by regulating the remodeling of junctional proteins and membrane-associated cytoskeleton.

Altrenogest during early pregnancy modulates uterine glandular epithelium and endometrial growth factor expression at the time implantation in pigs.

This study evaluated the effects of supplying altrenogest from day 6-12 of pregnancy on the endometrial glandular epithelium, corpora lutea (CL) morphology, and endometrial and CL gene expression. A total of 12 crossbred females (Landrace × Large White) were used. The females were assigned to 4 treatments according to a random design with a 2 × 2 factorial arrangement, with two categories (sow or gilt) and two treatments (non-treated and treated with altrenogest). On day 6 of pregnancy, animals were allocated to one of the following groups: non-treated (NT, n = 6; 3 sows and 3 gilts), and (T, n = 6; 3 sows and 3 gilts) treated daily with 20 mg of altrenogest, from day 6-12 of pregnancy. All animals were euthanized on day 13 of pregnancy. All CLs were individually weighed, and their volume were determined. The endometrial glandular density (GD), mean glandular area (MGA), and vascular density (VD) were determined by histomorphometric and immunohistochemical analyses. Endometrium samples were collected and analyzed by qRT-PCR to evaluate the abundance of transcripts for VEGF and IGF-I. Females in the T group had higher MGA (P < 0.05) compared to the NT group. There was no effect of treatment on GD or VD for both experimental groups. Sows in the T group had augmented expression of IGF-I (P < 0.05). Progestagen had no detrimental effect on CL morphology. In conclusion, altrenogest improves the uterine environment during the peri-implantation period in pigs without compromising corpora lutea development.

Cell-Specific Expression of Enzymes for Serine Biosynthesis and Glutaminolysis in Farm Animals.

During the peri-implantation period, conceptuses [embryo and placental membranes, particularly the trophectoderm (Tr)] of farm animals (e.g., sheep and pigs) rapidly elongate from spherical to tubular to filamentous forms. In concert with Tr outgrowth during conceptus elongation, the Tr of sheep and pig conceptuses attaches to the endometrial luminal epithelium (LE) to initiate placentation. In sheep, binucleate cells (BNCs) begin to differentiate from the mononuclear trophectoderm cells and migrate to the endometrial LE to form syncytial plaques. These events require Tr cells to expend significant amounts of energy to undergo timely and extensive proliferation, migration and fusion. It is likely essential that conceptuses optimally utilize multiple biosynthetic pathways to convert molecules such as glucose, fructose, and glutamine (components of histotroph transport by sheep and pig endometria into the uterine lumen), into ATP, amino acids, ribose, hexosamines and nucleotides required to support early conceptus development and survival. Elongating and proliferating conceptus Tr cells potentially act, in a manner similar to cancer cells, to direct carbon generated from glucose and fructose away from the TCA cycle for utilization in branching pathways of glycolysis, including the pentose phosphate pathway, one-carbon metabolism, and hexosamine biosynthesis. The result is alimited availability of pyruvate for maintaining the TCA cycle within mitochondria, and Tr cells replenish TCA cycle metabolites via a process known as anaplerosis, primarily through glutaminolysis to convert glutamine into TCA cycle intermediates. Here we describe the cell-specific expression of enzymes required for serine biosynthesis, one-carbon metabolism and glutaminolysis at the uterine-placental interface of sheep and pigs, and propose that these biosynthetic pathways are essential to support early placental development including Tr elongation, cell migration, cell fusion and implantation by ovine and porcine conceptuses.

Histopathological alterations due to different phylogroups of Escherichia coli isolated from clinical cases of bovine endometritis in rat model

In the present study, E. coli isolates representing four different phylogroups viz. A, B1, D, and F were used in rat model to study their pathogenic potential for the development of endometritis. Animal were classified into four infected groups and one group served as negative control. Isolates representing respective phylogroups were inoculated by intrauterine route in twenty diestrus rats. Histopathological changes noted in all the experimental groups with varying intensity of lesions of endometritis characterized by invasion of neutrophils in endometrial superficial epithelium, stratum compactum, uterine cavity, and microabscess formation. Experimental group 4 (phylogroup D) showed highest mean histopathological score, followed by group 2 (phylogroup A), group 3 (phylogroup B1) and group 5 (phylogroup F). However, no statistical significant difference could be observed between the infected groups. The present study concluded that, different phylogroups of E. coli do not differ significantly to induce the lesions of endometritis in diestrus rats.

Opaque Polyploid Cells in Ishikawa Endometrial Cultures Are Capable of Forming Megamitochondria, Organelles Derived from the Adaptation of Fused Mitochondria Whose Capacity to Develop Gaseous Vacuoles Suggests CO&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; Retention and Hypoxic Metabolism

Opaque polyploid cells capable of forming megamitochondria are a constant feature in colonies of Ishikawa endometrial epithelia, accounting for approximately 5% - 10% of the cells. Opaque cells appear to communicate with other opaque cells via membrane extensions and with other cells in a colony by extra-cellular vesicles. Opaque cells form first as rectangular structures, somewhat larger than surrounding monolayer cells. The cells eventually round up, remaining in the colony for 20 or more hours before detaching. The most unusual characteristic of Ishikawa opaque cells is their capacity to form mitonucleons, megamitochondria that surround aggregated chromatin. This paper reviews evidence that adaptations resulting in megamitochondria include a loss of the capacity for oxidative phosphorylation leaving the adapted megamitochondria reliant on metabolism such as reductive carboxylation.

Evaluation of glycan expression in endometrial epithelial structures as a predictive factor for endometrial receptivity

Aim. To evaluate the endometrial glyotype in the implantation window in patients with infertility in assisted reproductive technologies (ART) programs and to develop criteria for assessing endometrial receptivity based on the analysis of complex changes in the functional groups of glycans in the main endometrial epithelial to use in clinical practice.&#x0D; Materials and methods. The study included 32 infertile patients who took part in ART programs. Pipelle endometrial biopsy was performed during the implantation window in the cycle preceding the cycle of ovarian stimulation and embryo transfer. Using lectin histochemistry and immunohistochemistry, glycans levels in the glycocalyx of endometrial surface epithelium (SE) and glandular epithelium (GE) was studied.&#x0D; Results. Differences in the expression of mannose-rich glycans in SE and N-acetylgalactosamine--terminated glycans in the GE of the endometrium were revealed, as well as the link between the expression of lectin staining glycans: ConA, VVL, UEA-I, SNA, MAL-II, ECL and monoclonal antibodies: anti-MECA -79 mAb and anti-LeY mAb in SE and GE of the endometrium was assessed in patients with infertility, depending on the outcome of the ART program. The estimated threshold levels in glycan expression and the odds ratio for pregnancy occurrence allow to characterize the diagnostic value for the studied glycomarkers and to develop criteria for assessing endometrial receptivity.&#x0D; Conclusion. Based on a comprehensive evaluation of glycans in the main endometrial epithelial structures in the cycle preceding the ART cycle, criteria for assessment of the likelihood of pregnancy have been developed, which can increase the effectiveness of ART programs in patients with infertility.