Leukemia Inhibitory Factor Research Articles

Leukemia inhibitory factor shortens primary cilia by upregulating C-C motif chemokine 2 in human neural stem/progenitor cells.

Primary cilia on neural stem/progenitor cells (NSPCs) play an important role in determining cell fate, although the regulatory mechanisms involved in the ciliogenesis remain largely unknown. In this study, we analyzed the effect of the leukemia inhibitory factor (LIF) for the primary cilia in immortalized human NSPCs. LIF withdrawal elongated the primary cilia length, whereas the addition of LIF shortened it. Microarray gene expression analysis revealed that differentially expressed genes (DEGs) associated with LIF treatment were related with the multiple cytokine signaling pathways. Among the DEGs, C-C motif chemokine 2 (CCL2) had the highest ranking and its increase in the protein concentration in the NSPCs-conditioned medium after the LIF treatment was confirmed by ELISA. Interestingly, we found that CCL2 was a negative regulator of cilium length, and LIF-induced shortening of primary cilia was antagonized by CCL2-specific antibody, suggesting that LIF could influence cilia length via upregulating CCL2. The shortening effect of LIF and CCL2 on primary cilia was also observed in SH-SY5Y cells. The results of the study suggested that the LIF-CCL2 axis may well be a regulator of NSPCs and its primary cilia length, which could affect multiple cellular processes, including NSPC proliferation and differentiation.

Heterogeneity in the Metastatic Microenvironment: JunB-Expressing Microglia Cells as Potential Drivers of Melanoma Brain Metastasis Progression.

Reciprocal signaling between melanoma brain metastatic (MBM) cells and microglia reprograms the phenotype of both interaction partners, including upregulation of the transcription factor JunB in microglia. Here, we aimed to elucidate the impact of microglial JunB upregulation on MBM progression. For molecular profiling, we employed RNA-seq and reverse-phase protein array (RPPA). To test microglial JunB functions, we generated microglia variants stably overexpressing JunB (JunBhi) or with downregulated levels of JunB (JunBlo). Melanoma-derived factors, namely leukemia inhibitory factor (LIF), controlled JunB upregulation through Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling. The expression levels of JunB in melanoma-associated microglia were heterogeneous. Flow cytometry analysis revealed the existence of basal-level JunB-expressing microglia alongside microglia highly expressing JunB. Proteomic profiling revealed a differential protein expression in JunBhi and JunBlo cells, namely the expression of microglia activation markers Iba-1 and CD150, and the immunosuppressive molecules SOCS3 and PD-L1. Functionally, JunBhi microglia displayed decreased migratory capacity and phagocytic activity. JunBlo microglia reduced melanoma proliferation and migration, while JunBhi microglia preserved the ability of melanoma cells to proliferate in three-dimensional co-cultures, that was abrogated by targeting leukemia inhibitory factor receptor (LIFR) in control microglia-melanoma spheroids. Altogether, these data highlight a melanoma-mediated heterogenous effect on microglial JunB expression, dictating the nature of their functional involvement in MBM progression. Targeting microglia highly expressing JunB may potentially be utilized for MBM theranostics.

OR2803 Corticotroph Fate Weighted Cell Differentiation Paradigm For Human Induced Pluripotent Stem Cells

Abstract Disclosure: M.E. Moore: None. K.G. Chen: None. K. Park: None. D. Asuzu: None. N. Ramavenkat: None. S. Walbridge: None. D. Mullaney: None. M. Arhin: None. D. Maric: None. D. Mandal: None. P. Chittiboina: None. Introduction: The vertebrate anterior pituitary gland is a highly conserved ‘master’ organ that controls hormonal milieu in real-time. Pituitary adenomas and their treatments (surgery, chemotherapy or radiation) can result in pituitary organ failure, specifically hypocortisolism, a challenging condition to manage. Here, we created an optimized paradigm (anterior pituitary corticotroph or APcor) to preferentially drive human induced pluripotent stem cells (hiPSCs) towards a pituitary corticotroph fate. Methods: We directed U112I hiPSCs towards terminally differentiated cell types using a combination of rho-associated kinase inhibitor (Y-27632), bone morphogenic protein 4 (BMP4), transforming growth factor β inhibitor (SB431542), sonic hedgehog (SHH), fibroblast growth factor 8b (FGF8b), fibroblast growth factor 10 (FGF10) and leukemia inhibitory factor (LIF). Markers for pluripotent stem cells, cranial placode (CP), Rathke’s Pouch (RP), anterior pituitary progenitors and mature cell types were assessed at 10-day intervals using quantitative qRT-PCR and multiplex immunocytochemistry (mICC) or immunohistochemistry (IHC). ACTH activity was measured using ELISA (MD Bio). Results: We tested initial differentiation conditions and found optimal CP initiation in presence of SB431542. We observed decrease in embryonic stem cell markers OCT4 and NANOG by day 10, with concomitant increase in CP/RP markers SIX1, PAX6 and SIX6 using mICC/IHC and qRT-PCR. Continuation of differentiation paradigm with LIF showed strong expression of anterior pituitary progenitor markers PITX1, TBX19 and POU1F1 at day 20. pRT-PCR analysis at this time point confirms differentiation towards pituitary cell types without needing enrichment for SIX1. At day 30, POMC expression was observed by IHC in 10-15% of cells as measured by quantitative analysis (Qupath 0.2.0), indicating mature corticotrophs. Corticotroph cell fate was confirmed with corticotropin releasing hormone (CRH) stimulation which led to up to 40x-fold increase in ACTH expression detected by ELISA (D30 w/CRH vs Pluripotent w/CRH). Conclusions: We found with the APcor paradigm, we can reliably drive towards corticotroph cell fate. We successfully differentiated hiPSCs into mature functioning corticotrophs with ability to recapitulate pituitary organ function. Our method is amenable to 3D organoid formation and pre-clinical implantation in animal models of hypophysectomy as a novel hormone replacement therapy. Presentation: Sunday, June 18, 2023

STAT3-dependent long non-coding RNA Lncenc1 contributes to mouse ES cells pluripotency via stabilizing Klf4 mRNA.

Embryonic stem cells (ESCs) preserve the unique ability to differentiate into any somatic cell lineage while maintaining their self-renewal potential, relying on a complex interplay of extracellular signals regulating the expression/activity of pluripotency transcription factors and their targets. Leukemia inhibitory factor (LIF)-activated STAT3 drives ESCs' stemness by a number of mechanisms, including the transcriptional induction of pluripotency factors such as Klf4 and the maintenance of a stem-like epigenetic landscape. However, it is unknown if STAT3 directly controls stem-cell specific non-coding RNAs, crucial to balance pluripotency and differentiation. Applying a bioinformatic pipeline, here we identify Lncenc1 in mouse ESCs as an STAT3-dependent long non-coding RNA that supports pluripotency. Lncenc1 acts in the cytoplasm as a positive feedback regulator of the LIF-STAT3 axis by competing for the binding of microRNA-128 to the 3'UTR of the Klf4 core pluripotency factor mRNA, enhancing its expression. Our results unveil a novel non-coding RNA-based mechanism for LIF-STAT3-mediated pluripotency.

Efficacy of the long-acting gonadotropin-releasing hormone agonist long protocol on IVF/ICSI outcomes of patients with repeated implantation failure.

The present study aimed to investigate the effect of long-acting gonadotropin-releasing hormone agonist (GnRHa) long protocol on in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes of patients with repeated implantation failure (RIF). The present study was carried out from June 1, 2016 to June 30, 2021. A total of 665 patients with RIF were enrolled into the study and classified by the ovarian stimulation protocols. The outcome parameters were compared in each group. In addition, we evaluated the expression of homeobox A10 (HOXA10), integrin β3 and leukemia inhibitory factor (LIF) in endometrial tissues between groups by quantitative RT-PCR. Patients who received the long-acting GnRHa long protocol had significantly higher clinical pregnancy rates (58.0%, 41.7% and 39.9%, respectively; P = 0.008 and 0.003), implantation rates (38.1%, 30.3%, and 30.1%, respectively; P = 0.001 and <0.001) and live birth rates (50.3%, 36.3%, and 31.3%, respectively; P = 0.020 and 0.002) compared with the short-acting GnRHa long protocol and GnRH antagonist protocol. In addition, we found that long-acting GnRHa could improve the expression of HOXA10 (P < 0.05). The long-acting GnRHa long protocol could improve endometrial receptivity and IVF/ICSI clinical outcomes of RIF patients compared with the short-acting GnRHa long protocol and GnRH antagonist protocol.

Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, Cardiotrophin-1 Expression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration.

Persistent oxidative stress and inflammation contribute causally to smooth muscle cell (SMC) proliferation and migration, the characteristic features of vascular proliferative diseases. Oxidatively modified low-density lipoproteins (OxLDL) elevate oxidative stress levels, inflammatory responses, and matrix metallopeptidase (MMP) activation, resulting ultimately in SMC migration, proliferation, and phenotype change. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Empagliflozin is an SGLT2 inhibitor and exerts pleiotropic cardiovascular protective effects, including antioxidant and anti-inflammatory effects. Here, we investigated (i) whether OxLDL regulates RECK expression, (ii) whether ectopic expression of RECK reverses OxLDL-induced SMC migration and proliferation, and (iii) whether pretreatment with empagliflozin reverses OxLDL-induced RECK suppression, MMP activation, and SMC migration, proliferation, and differentiation. Indeed, results show that OxLDL at pathophysiological concentration promotes SMC migration and proliferation via NF-κB/miR-30b-dependent RECK suppression. Moreover, OxLDL changed the SMC phenotype to a more pro-inflammatory type, and this effect is blunted by RECK overexpression. Further, treatment with empagliflozin reversed OxLDL-induced miR-30b induction, RECK suppression, MMP activation, SMC migration, proliferation, and proinflammatory phenotype changes. OxLDL-induced cardiotrophin (CT)-1 expression and CT-1 stimulated SMC proliferation and migration in part via leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130). Ectopic expression of RECK inhibited these effects by physically associating with LIFR and gp130, as evidenced by immunoprecipitation/immunoblotting and double immunofluorescence. Importantly, empagliflozin inhibited CT-1-induced mitogenic and migratory effects. Together, these results suggest the therapeutic potential of sustaining RECK expression or empagliflozin in vascular diseases characterized by SMC proliferation and migration.

LIF Aggravates Pulpitis by Promoting Inflammatory Response in Macrophages.

Leukemia inhibitory factor (LIF) has been recognized as a novel inflammatory modulator in inflammation-associated diseases. This study aimed to investigate the modulation of LIF in dental pulp inflammation. Experimental pulpitis was established in wild-type (WT) and Lif-deficient (Lif-/-) mice. Histological and immunostaining analyses were conducted to assess the role of LIF in the progression of pulpitis. Mouse macrophage cell line (RAW264.7) was treated with LPS to simulate an inflammatory environment. Exogenous LIF was added to this system to examine its modulation in macrophage inflammatory response in vitro. Primary bone marrow-derived macrophages (BMDMs) from WT and Lif-/- mice were isolated and stimulated with LPS to confirm the effect of Lif deletion on macrophage inflammatory response. Supernatants from LIF and LPS-treated human dental pulp cells (hDPCs) were collected and added to macrophages. Macrophage chemotaxis was assessed using transwell assays. The results showed an increased expression of LIF and LIFR with the progression of pulpitis, and LIFR was highly expressed in macrophages. Lif deficiency alleviated experimental pulpitis with the reduction of pro-inflammatory cytokines and macrophage infiltration. Exogenous LIF promoted inflammatory response of LPS-induced macrophages through a STAT3/p65-dependent pathway. Consistently, Lif deletion inhibited macrophage inflammatory response in vitro. Supernatants of LIF-treated hDPCs enhanced macrophage migration in LPS-induced inflammatory environment. Our findings demonstrated that LIF aggravates pulpitis by promoting macrophage inflammatory response through a STAT3/p65-dependent pathway. Furthermore, LIF plays a crucial role in driving the recruitment of macrophages to inflamed pulp tissue by promoting chemokine secretion in DPCs.

LIF signaling regulates outer radial glial to interneuron fate during human cortical development.

Radial glial (RG) development is essential for cerebral cortex growth and organization. In humans, the outer radial glia (oRG) subtype is expanded and gives rise to diverse neurons and glia. However, the mechanisms regulating oRG differentiation are unclear. oRG cells express leukemia-inhibitory factor (LIF) receptors during neurogenesis, and consistent with a role in stem cell self-renewal, LIF perturbation impacts oRG proliferation in cortical tissue and organoids. Surprisingly, LIF treatment also increases the production of inhibitory interneurons (INs) in cortical cultures. Comparative transcriptomic analysis identifies that the enhanced IN population resembles INs produced in the caudal ganglionic eminence. To evaluate whether INs could arise from oRGs, we isolated primary oRG cells and cultured them with LIF. We observed the production of INs from oRG cells and an increase in IN abundance following LIF treatment. Our observations suggest that LIF signaling regulates the capacity of oRG cells to generate INs.

Circulating biomarkers at diagnosis correlate with distant metastases of early luminal-like breast cancer

There is an urgent need for new and better biomarker modalities to estimate the risk of recurrence within the luminal-like breast cancer (BC) population. Molecular diagnostic tests used in the clinic lack accuracy in identifying patients with early luminal BC who are likely to develop metastases. This study provides proof of concept that various liquid biopsy read-outs could serve as valuable candidates to build a multi-modal biomarker model distinguishing, already at diagnosis, between early metastasizing and non-metastasizing patients. All these blood biomarkers (chemokines, microRNAs, leukemia inhibitory factor, osteopontin, and serum-induced functional myeloid signaling responses) can be measured in baseline plasma/serum samples and could be added to the existing prognostic factors to improve risk stratification and more patient-tailored treatment in early luminal BC.

Electroacupuncture combined with bone marrow mesenchymal stem cell transplantation promotes repair of thin endometrium by regulating SDF-1/CXCR4 signaling

It is to explore, based on stromal cell derived factor 1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signal axis, whether the electroacupuncture (EA) combined with bone marrow mesenchymal stem cells (BMSCs) transplantation can promote thin endometrium regeneration and improve endometrial receptivity, so as to further study its mechanisms underlying improvement of promoting BMSCs homing to repair thin endometrium. Thirty matured female SD rats were randomly divided into normal control , model , BMSCs transplantation (BMSCs), BMSCs+AMD3100 (a specific antagonist of CXCR4, BMSCs+AMD3100), BMSCs+EA, and BMSCs+EA+AMD3100 groups, with 5 rats in each group. The thin endometrial model was established by intrauterine injection of 95% ethanol during the period of estrus. Rats of the model group received intravenous injection of PBS solution (tail vein) on day 1, 3 and 7 of modeling and intraperitoneal injection of normal saline once daily for 3 estrous cycles. Rats of the BMSCs group received intravenous injection of BMSCs suspension on day 1,3 and 7 of modeling, and those of the BMSCs+EA group received BMSCs transplantation and EA stimulation. EA (2 Hz/15 Hz, 1 mA) was applied to "Guanyuan" (CV4) and bilateral "Sanyinjiao"(SP9), "Zigong" (EX-CA1) for 15 min, once daily for 3 estrous cycles. Rats of the BMSCs+AMD3100 group received intravenous injection of BMSCs suspension (1×106/mL) and intraperitoneal injection of AMD3100 (5 mg/kg), and those of the BMSCs+EA+AMD3100 group received administration of BMSCs, AMD3100 and EA, with both groups being once daily for 3 estrous cycles. H.E. staining was used to observe histopathological changes of endometrium tissues, and immunohistochemistry was used to detect the expressions of cytokeratin (CK19) and vimentin in endometrium (for evaluating the damage and repair of endometrium). The expression levels of homeobox A10 (HOXA10), leukemia inhibitory factor (LIF), SDF-1 and CXCR4 proteins were detected by Western blot, and those of SDF-1 and CXCR4 mRNAs in the endometrium detected by real-time PCR. In comparison with the normal control group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression leve-ls of HOXA10, LIF and CXCR4 proteins and CXCR4 mRNA were significantly down-regulated (P<0.01), and the expression levels of SDF-1 protein and mRNA significantly up-regulated (P<0.05) in the model group. Compared with the model group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, and the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs group, and the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA, and SDF-1 protein and mRNA in the BMSCs+EA group were significantly up-regulated (P<0.05, P<0.01). Compared to the BMSCs group, the number of endometrial glands, and the expression levels of LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA group were up-regulated (P<0.01, P<0.05)； the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+AMD3100 group were down-regulated (P<0.01). Compared to the BMSCs+EA group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA+AMD3100 group were down-regulated (P<0.01). Results of H.E. staining showed thin endometrium with absence of epithelial cells, and sparse glands and blood vessels, with smaller glandular cavity in the model group, which was relative milder in BMSCs and BMSCs+EA groups. EA can promote the transfer of transplanted BMSCs to the damaged site through SDF-1/CXCR4 signaling related stem cell homing, thereby promoting thin endometrial regeneration, repairing endometrial injury, and improving endometrial tolerance in rats with thin endometrium.

Exploring the Potential of Azo Compounds in Leukemia Treatment: Synthesis and Characterization of New Derivatives with Dimedone and Meldrum's Acid End Groups

AbstractThis scholarly investigation presents pioneering drug candidates for combating leukemia, distinguished by reduced side effects and elevated efficacy. The study focuses on azo compounds for their selective targeting against cancerous cells, and customizable properties. Novel azo compounds, DMTPC and DMTPD, were skillfully synthesized via diazotization of 3‐(4‐Methyl‐1H‐imidazol‐1‐yl)‐5‐(trifluoromethyl)aniline with dimedone and meldrum‘s acid. Comprehensive characterization employed advanced analytical techniques like 1H‐NMR, 13C‐NMR, LC‐MS, FT‐IR, and XRD. UV‐Vis and fluorescence spectrophotometers scrutinized absorption and fluorescence properties in diverse solvents. Theoretical DFT computations provided optimized geometries, vibrational frequencies, NMR chemical shifts, absorption wavelengths, HOMOs‐LUMOs, Mulliken charges, and molecular orbital energies. Encouragingly, experimental and theoretical values aligned well. ADME properties and toxicity profiles were evaluated using online web servers. Molecular dynamics simulations explored interactions of DMTPC and DMTPD with the leukemia inhibitory factor protein (PDB ID: 1EMR) compared to Nilotinib. Molecular docking studies indicated DMTPC′s promising potential with a binding energy of −8.8 kcal/mol, approaching Nilotinib's −9.4 kcal/mol. This investigation accentuates the burgeoning research paradigm of exploiting azo compounds in the formidable battle against leukemia, opening new avenues for breakthroughs in this field.

Study the effect of recombinant leukemia inhibitory factor on maintenance of pregnancy and frequency of regulatory T cells in abortion-prone mice

Recurrent spontaneous abortion (RSA) can have a significant impact on a woman's quality of life. Understanding the mechanisms behind abortion is crucial for developing potential treatments. Among various models of abortion, the CBA/J(♀) × DBA/2J(♂) model stands out as the most extensively studied. This model reveals the influence of an altered immune system on resorption during pregnancy. The leukemia inhibitory factor (LIF) holds considerable importance as a secretory glycoprotein essential for successful implantation. Regulatory T cells (Tregs) have been found to produce high levels of LIF in both mice and humans. LIF plays a vital role in the development of Tregs by upregulating the expression of the Foxp3 transcription factor while downregulating the expression of RORγt. To investigate the impact of recombinant LIF (rLIF) on pregnancy maintenance and Treg cell frequency in abortion-prone (AP) mice, a specific recombinant protein was used in this study. The AP group consisted of CBA/J(♀) × DBA/2J(♂) mice, while the control group comprised CBA/J(♀) × BALB/c(♂) mice. Intraperitoneal injections of rLIF were administered to the AP group on the third day of pregnancy, and its effects on Treg cell frequency and pregnancy maintenance were examined during this period. Following rLIF injections on the fourteenth day of pregnancy, the expression of Foxp3 significantly increased in AP mice (p = 0.02,0.008). Additionally, AP mice injected with rLIF demonstrated a significant reduction in resorption rate (p = 0.01) and a notable increase in birth rate (p = 0.01,0.0005). These findings provide new insights into the potential benefits of LIF in treating RSA patients.

Endometrium procurement and transplantation restores fertility in rats

Research questionCan rat endometrium be successfully procured and transplanted, and can a similar method be used to procure human endometrium? DesignRat endometrium was procured using an endometrium stripping method and transplanted into female Sprague–Dawley rats. Macroscopic and histological changes, endometrial receptivity-related protein concentrations and fertility were assessed. Additionally, a preliminary experiment was conducted to procure human endometrium using a similar method. ResultsEndometrium was successfully procured from both rats and humans, which contained intact endometrium and parts of the adjacent inner annulus myometrium. Endometrium auto-transplantation was conducted in rats and the procedure lasted a total of 41.3 ± 5.7 min with a mean blood loss of 0.09 ± 0.04 g. The transplanted endometrium survived well, but a fibrotic zone formed between the transplant and recipient tissue. Compared with sham rats, those with endometrium transplantation had similar endometrial thickness and endometrial gland numbers but reduced vascular density at 8 weeks after surgery. Endometrium transplantation also retained expression of the endometrial receptivity-related proteins leukaemia inhibitory factor and vascular endothelial growth factor. In contrast to non-pregnancy in the stripped horn, a mean of 5.0 ± 2.7 fetuses developed in the transplanted horn, and full-term live fetuses were conceived in the horns with transplanted endometrium. ConclusionsEndometrium procurement by stripping method can obtain an intact and functional endometrium, and endometrium transplantation can reconstruct the uterine cavity and restore fertility in rats.

Cancer Stem Cells in Renal Cell Carcinoma: Origins and Biomarkers.

The term "cancer stem cell" (CSC) refers to a cancer cell with the following features: clonogenic ability, the expression of stem cell markers, differentiation into cells of different lineages, growth in nonadhesive spheroids, and the in vivo ability to generate serially transplantable tumors that reflect the heterogeneity of primary cancers (tumorigenicity). According to this model, CSCs may arise from normal stem cells, progenitor cells, and/or differentiated cells because of striking genetic/epigenetic mutations or from the fusion of tissue-specific stem cells with circulating bone marrow stem cells (BMSCs). CSCs use signaling pathways similar to those controlling cell fate during early embryogenesis (Notch, Wnt, Hedgehog, bone morphogenetic proteins (BMPs), fibroblast growth factors, leukemia inhibitory factor, and transforming growth factor-β). Recent studies identified a subpopulation of CD133+/CD24+ cells from ccRCC specimens that displayed self-renewal ability and clonogenic multipotency. The development of agents targeting CSC signaling-specific pathways and not only surface proteins may ultimately become of utmost importance for patients with RCC.

Receptivity of the endometrium in infertility associated with adenomyosis

Violation of the receptivity of the endometrium in adenomyosis can be supported either by pathological processes in the structure of the endometrium, such as an unexpected increase in vascularization and thickness of the endometrial stroma with a violation of the endometrial environment, or a negative effect on implantation, or a change in the expression of enzymes, cytokines, growth factors, adhesion molecules and various genes, such as hypoxia-induced factor 1α, interleukins (IL-6, IL-8, IL-10, IL-II), IL-8 receptors, CXCR1, CXCR2, matrix metalloproteinases (MMP2 and MMP9), vascular endothelial growth factor, leukemia inhibitory factor (LIF), LIF receptor α, HOXA10 gene, cytochrome P450, aromatase activity, estrogen receptor α, cell adhesion molecules such as integrins, MUC1, pinopods and L-selection. The modern literature data on the receptivity of the endometrium in infertility associated with adenomyosis was analyzed, the etiology and morphofunctional features of the endometrium, its implantation disorders as well as their effects on implantation processes were studied. Infertility is one of the most important and complex medical and social problems in the modern world. Thanks to advances in imaging techniques, there is now increasing evidence that adenomyosis is one of the causes of female infertility. According to studies, adenomyosis was observed in 27% of infertile patients treated with assisted reproductive technologies (ART). Adenomyosis is a disease with an unclear etiology, the pathogenesis of the disease identifies several theories, but there is no single correct one, its connection with infertility in women requires further study and depends on many factors, one of which is the receptivity of the endometrium. Treatment of this disease requires a personalized approach for each patient, to achieve the main goal of overcoming infertility.

Effect of tetrabromobisphenol A (TBBPA) on early implantation using the three-dimensional spheroid model with human endometrial cell line, Ishikawa

BackgroundTetrabromobisphenol A (TBBPA) can be characterized as an endocrine-disrupting chemical (EDCs). It has been widely used as a brominated flame retardant in industrial products. EDCs have effects on female reproduction leading to issues, such as infertility, hormone imbalance, and endometriosis. In Korea, the problems of infertility and decreasing birth rate are of significant concern. Exposure to EDCs might have a harmful effect on female fertility by mediating a decrease endometrial receptivity. This study aimed to investigate the effects of TBBPA on infertility, particularly on early implantation events in the uterine endometrium. Human endometrial adenocarcinoma and trophoblastic cell lines were used in this study. The cytotoxicity of TBBPA on Ishikawa cells and Jeg-3 cells was measured using the Cell Counting Kit-8 assay. The mRNA expression was analyzed by reverse transcription-quantitative polymerase chain reaction, and protein levels were measured by western blotting. The attachment rate was analyzed using an attachment assay, and the outgrowth area was measured using an outgrowth assay.ResultsThe mRNA expression of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and leukemia inhibitory factor was significantly increased upon treatment of Ishikawa cells by TBBPA. Moreover, the outgrowth area in the TBBPA group was significantly decreased compared to that in the control. In contrast, TBBPA had a minor effect on protein levels and attachment rates.ConclusionsIn this study, TBBPA induced an inflammatory milieu in mRNA expression. An increase in inflammation-related cytokines in the endometrium can disrupt embryo implantation. TBBPA disrupted the outgrowth of spheroids in the endometrium; however, the protein levels and attachment rate were comparable to those in the control group. The effect of TBBPA on implantation events should be elucidated further.

AUF1-induced circular RNA hsa_circ_0010467 promotes platinum resistance of ovarian cancer through miR-637/LIF/STAT3 axis.

Increasing evidences has indicated that primary and acquired resistance of ovarian cancer (OC) to platinum is mediated by multiple molecular and cellular factors. Understanding these mechanisms could promote the therapeutic efficiency for patients with OC. Here, we screened the expression pattern of circRNAs in samples derived from platinum-resistant and platinum-sensitive OC patients using RNA-sequencing (RNA-seq). The expression of hsa_circ_0010467 was validated by Sanger sequencing, RT-qPCR, and fluorescence in situ hybridization (FISH) assays. Overexpression and knockdown experiments were performed to explore the function of hsa_circ_0010467. The effects of hsa_circ_0010467 on enhancing platinum treatment were validated in OC cells, mouse model and patient-derived organoid (PDO). RNA pull-down, RNA immunoprecipitation (RIP), and dual-luciferase reporter assays were performed to investigate the interaction between hsa_circ_0010467 and proteins. Increased expression of hsa_circ_0010467 is observed in platinum-resistant OC cells, tissues and serum exosomes, which is positively correlated with advanced tumor stage and poor prognosis of OC patients. Hsa_circ_0010467 is found to maintain the platinum resistance via inducing tumor cell stemness, and silencing hsa_circ_0010467 substantially increases the efficacy of platinum treatment on inhibiting OC cell proliferation. Further investigation reveals that hsa_circ_0010467 acts as a miR-637 sponge to mediate the repressive effect of miR-637 on leukemia inhibitory factor (LIF) and activates the LIF/STAT3 signaling pathway. We further discover that AUF1 could promote the biogenesis of hsa_circ_0010467 in OC. Our study uncovers the mechanism that hsa_circ_0010467 mediates the platinum resistance of OC through AUF1/hsa_circ_0010467/miR-637/LIF/STAT3 axis, and provides potential targets for the treatment of platinum-resistant OC patients.

The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity

ABSTRACT The mammalian endometrium is covered by the lumenal epithelium (Le), which directly interacts with the blastocyst and plays an important role in the establishment of reciprocal crosstalk between the embryo and receptive uterus during implantation. However, the effect of the blastocyst on uterine differentiation during the window of receptivity is far from well understood. Through transcriptomic profiling of the uterine Le isolated by laser capture microdissection (LCM), it was demonstrated that global gene expression changes occurred in Le between pseudopregnant mice without embryos and pregnant mice with embryos. Some differentially expressed genes, including upregulated Areg (amphiregulin), Ihh (Indian hedgehog), Lifr (leukemia inhibitory factor receptor) and downregulated Msx1 (msh homeobox 1), Pgr (progesterone receptor), and Gata2 (GATA binding protein 2) in pregnant mice, have been reported to regulate the establishment of uterine receptivity. Besides, we found that blastocysts induced an increase in both the number and acidification of lysosome, consistent with enhanced lysosomal hydrolase activity in uterine Le. Further exploration uncovered that blastocyst-derived IGF2 was involved into the activation of epithelial STAT3 to induce lysosomal hydrolase expression, and inhibition of lysosomal function derails both uterine receptive maker gene expressions and embryo implantation. Finally, based on the proteomic data of both epithelia and the separated lysosome, it was revealed that CLDN1 (claudin 1) and MUC1 (mucin 1, transmembrane), two well-known downregulated molecules for successful implantation, are degraded by epithelial lysosome. In brief, our data demonstrated that blastocysts induced normal epithelium differentiation with lysosome activation to promote the uterine epithelial differentiation for embryo implantation. Abbreviations ACTB: actin beta; AREG: amphiregulin; ATP6V0A4: ATPase, H+ transporting, lysosomal V0 subunit A4; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CLDN1: claudin 1; CTSB: cathepsin B; DEGs: differentially expressed genes; E2: 17β-estradiol; ESR: estrogen receptor; GATA2: GATA binding protein 2; GLA: galactosidase, alpha; GO: gene ontology; HBEGF: heparin-binding EGF-like growth factor; IGF1R: insulin-like growth factor 1 receptor; Ihh: Indian hedgehog; ISH: in situ hybridization; LAMP1: lysosomal-associated membrane protein 1; LCM: laser capture microdissection; Le: lumenal epithelium; LGMN: legumain; LIF: leukemia inhibitory factor; LIFR: LIF receptor alpha; MSX1: msh homeobox 1; MUC1: mucin 1, transmembrane; P4: progesterone; PBS: phosphate-buffered saline; PCA: principal component analysis; PPT1: palmitoyl-protein thioesterase 1; PGR: progesterone receptor; PSP: pseudopregnancy; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; qPCR: quantitative real-time polymerase chain reaction; SP: pregnancy; TFEB: transcription factor EB.

An interplay of Progesterone, Leukemia Inhibitor Factor and Interleukin-6 in the window of implantation; Impact on fertility

BackgroundThe process of implantation is crucial for the initiation of conception and hence fertility. In addition to a number of factors, it is regulated by a cross talk of gonadotrophins [Luteinizing Hormone (LH), Follicle Stimulatory Hormone (FSH)], ovarian steroids [Estrogen (Et), Progesterone (Pt)] and cytokines [Leukemia inhibitory factor (LIF) and Interleukin 6 (IL6)]. These biomarkers are chief players of implantation. ObjectiveWe aimed to explore the role of gonadotrophins (LH, FSH, LH/FSH ratio), ovarian steroids (Et, Pt) and cytokines (LIF, IL6) in the implantation process. This aim was achieved by comparing these hormones and cytokines in the fertile and infertile groups [Polycystic ovaries (PCOs), endometriosis, unexplained infertility (Uex-IF)] and finding their association in all study groups. MethodsA case control study conducted from October 2020-March 2023. A total of 135 infertile women (with PCOs, Uex-IF, and endometriosis) and 177 fertile women (matched for age and BMI) were selected. Levels of ‘Et’, ‘Pt’, ‘LIF’ and, ‘IL6′ were estimated using Enzyme Linked Immunosorbent Assay (ELISA). LH and FSH values were obtained from hospital desk records.The Independent Student'st-test was used to compare fertile and infertile groups. One-way ANOVA test was used to compare more than two groups, and Pearson’s chi-square (χ2) test was employed to compare percentages of variables. Pearson correlation analysis was performed to assess the associations and correlations. A p value < 0.05 was considered statistically significant. ResultsSignificantly higher levels of LIF and IL6 were observed in fertile women compared to infertile women. Pt levels were significantly greater in the fertile group than in the infertile group. The FSH/LH ratio was significantly higher in the fertile group. Among infertile women, PCOs (71%) and Uex-IF (91%) exhibited lower Pt levels than the fertile controls (p < 0.01), but these levels remained within the reference range (RR). Among the fertile group (81%), levels of LIF within the RR were significantly higher compared to those with Uex-IF (49%) and females with endometriosis (37%). Moreover, the highest number of participants (57%) with Uex-IF exhibited IL6 levels significantly below the RR in comparison to the fertile group and infertile groups (PCOS and endometriosis). However, lower levels of IL6 were observed in women with Uex-IF.In the control group, LIF exhibited a significant positive correlation with IL6 (r = 0.370), Pt (r = 0.496), Et (r = 0.403), and LH (r = 0.428). Among women with PCOs, LIF showed a significant positive correlation with IL6 (r = 0.443), Pt (r = 0.607), and LH (r = 0.472). In cases of Uex-IF, LIF demonstrated a significant positive correlation with IL6 (r = 0.727). Females with endometriosis displayed a significant positive correlation between LIF and IL6 (r = 0.535) as well as Pt (r = 0.605). In fertile women, a positive correlation was observed between LH and IL6 (r = 0.197, p = 0.009), LIF (r = 0.428, p = 0.000), Pt (r = 0.238, p = 0.001), and Et (r = 0.356, p = 0.000). Furthermore, a positive correlation was found between LH and LIF (r = 0.472, p = 0.000) in women with PCOs. ConclusionElevated levels of Pt were found to increase the production of LIF in fertile females. However, infertile females with PCOs and Uex-IF exhibited deficient levels of Pt, supporting its role as a biomarker for successful implantation in infertile women. These females showed decreased levels of gonadotropins as well as reduced LH/FSH ratio and diminished secretion of receptivity marker LIF, in addition to reduced Pt secretion. This suggests that reduced gonadotropin levels contribute to a lower LH/FSH ratio, resulting in decreased Pt secretion and ultimately leading to low levels of LIF, thereby causing impaired implantation in women with PCOs and Uex-IF. The exploration of low levels of LIF in patients with endometriosis requires further investigation. The significantly low levels of IL6 in the Uex-IF group elucidate the role of this cytokine in association with decreased Pt and LIF synthesis within this group.

The Trophoblast Compartment Helps Maintain Embryonic Pluripotency and Delays Differentiation towards Cardiomyocytes.

Normal developmental progression relies on close interactions between the embryonic and extraembryonic lineages in the pre- and peri-gastrulation stage conceptus. For example, mouse epiblast-derived FGF and NODAL signals are required to maintain a stem-like state in trophoblast cells of the extraembryonic ectoderm, while visceral endoderm signals are pivotal to pattern the anterior region of the epiblast. These developmental stages also coincide with the specification of the first heart precursors. Here, we established a robust differentiation protocol of mouse embryonic stem cells (ESCs) into cardiomyocyte-containing embryoid bodies that we used to test the impact of trophoblast on this key developmental process. Using trophoblast stem cells (TSCs) to produce trophoblast-conditioned medium (TCM), we show that TCM profoundly slows down the cardiomyocyte differentiation dynamics and specifically delays the emergence of cardiac mesoderm progenitors. TCM also strongly promotes the retention of pluripotency transcription factors, thereby sustaining the stem cell state of ESCs. By applying TCM from various mutant TSCs, we further show that those mutations that cause a trophoblast-mediated effect on early heart development in vivo alter the normal cardiomyocyte differentiation trajectory. Our approaches provide a meaningful deconstruction of the intricate crosstalk between the embryonic and the extraembryonic compartments. They demonstrate that trophoblast helps prolong a pluripotent state in embryonic cells and delays early differentiative processes, likely through production of leukemia inhibitory factor (LIF). These data expand our knowledge of the multifaceted signaling interactions among distinct compartments of the early conceptus that ensure normal embryogenesis, insights that will be of significance for the field of synthetic embryo research.