Endometrial Cell Proliferation Research Articles

Platelet-rich fibrin improves repair and regeneration of damaged endometrium in rats.

Intrauterine adhesion (IUA) is the most common cause of uterine infertility. This study aims to evaluate whether platelet-rich fibrin (PRF) treatment can stimulate damaged endometrium regeneration in rats. First, hematoxylin and eosin (HE) staining, scanning and transmission electron microscopy, and ELISAs were used to evaluate the microstructure of PRF. Then, mechanical damage was used to establish an IUA rat model. A total of 40 SD female rats were randomized to three groups: PRF transplantation group, IUA group, and sham group. Rats were sacrificed at 3, 7, and 14 days and uteruses were obtained for further analysis. Finally, functional and histological recovery of the damaged endometrium was analyzed by pregnancy test, HE staining, Masson's staining, and immunohistochemistry. PRF has two distinct zones, platelets and fibrin zone. Long and narrow fibrin fibers interconnected with each other and formed a three-dimensional, flexible, and elastic structure; platelet aggregates were trapped in fibrin fibers, and each platelet is associated with several fibrin fibers. PRF exudates promoted endometrial stromal cell proliferation and migration in vitro. PRF transplantation was beneficial for maintaining uterine structure, promoting endometrial luminal epithelium and endometrial gland regeneration, and decreasing fibrotic areas in vivo. Intrauterine administration of PRF was demonstrated to be effective in preventing IUA and stimulating damaged endometrium regeneration in rats. This study not only provided a promising method for its potential in endometrial regeneration in women who suffer from uterine infertility but also may prevent IUA after intrauterine surgery in clinical cases.

β-Sitosterol Inhibits The Proliferation of Endometrial Cells via Regulating Smad7-Mediated TGF-β/Smads Signaling Pathway.

To investigate the effect of β-sitosterol on endometrial cells to understand the underlying mechanism. This is a laboratory-based experimental study conducted on animals and cells. Histological assays were performed to determine the effect of β-sitosterol on endometrial cells. The CCK-8 assay was used to assess the inhibitory effect of β-sitosterol on the proliferation of ectopic endometrial stromal cells (hEM15A). Flow cytometry was performed to evaluate the induction of apoptosis by β-sitosterol in hEM15A cells. The transwell invasion assay was conducted to measure the suppression of hEM15A cell migration by β-sitosterol. Western blot analyses were performed to analyze the effect of β-sitosterol on the expression of Smad family member 7 (Smad7) and the activity of transforming growth factor-β (TGF-β1), as well as the phosphorylation of Smad2 and Smad3. Histological assays showed that β-sitosterol regulates histopathology and induces apoptosis of endometrial cells in vivo. The CCK-8 assay revealed that β-sitosterol could inhibit the proliferation of hEM15A in human endometriosis patients. Flow cytometry showed that apoptosis was triggered by β-sitosterol in hEM15A. The transwell invasion assay indicated that the hEM15A migration under the β-sitosterol treatment group was suppressed. Western blot analyses suggested that β-sitosterol increased the expression of Smad7, decreased the activity of TGF-β1, and reduced the phosphorylation of Smad2 and Smad3. The effect of β-sitosterol was weakened by the silence of Smad7. The results suggest that β-sitosterol can inhibit the proliferation of endometrial cells and relieve endometriosis by inhibiting TGF-β-induced phosphorylation of Smads through regulation of Smad7.

LIF regulates the expression of miR-27a-3p and HOXA10 in bovine endometrial epithelial cells via STAT3 pathway

LIF is crucial in regulating embryo implantation, while HOXA10 is a marker gene for uterine receptivity. However, the specific mechanism of LIF regulating HOXA10 during cow embryo implantation has not been fully understood. To address this knowledge gap, the experiment involved treating bovine endometrial epithelial cells (BEECs) with LIF to investigate the relationship between LIF, miRNA, and HOXA10. The experimental findings revealed that applying LIF resulted in a substantial increase in the proliferation of endometrial epithelial cells. Moreover, the expressions of PI3K, AKT, HOXA10, CDK4, cyclinD1, and cyclinE1 were significantly elevated. Conversely, the expression of p21Cipl was significantly reduced. In the group that received a combination of LIF and a STAT3 inhibitor, the expression of PI3K/AKT remained significantly increased, but there was no significant change in the expression of HOXA10. When miRNA-27a-3p was overexpressed, it resulted in a decrease in both the RNA and protein expression of HOXA10. Conversely, inhibiting miRNA-27a-3p increased the RNA and protein expression of HOXA10. In the presence of LIF treatment, the expression of miRNA-27a-3p was reduced, while the expression of HOXA10 was increased. However, when LIF and a STAT3 inhibitor were combined, there was no significant change in the expression of miRNA-27a-3p or HOXA10. Consequently, LIF facilitated cell proliferation by activating the PI3K/AKT pathway. LIF controlled the expression of miRNA-27a-3p and HOXA10 in endometrial epithelial cells through STAT3, with miRNA-27a-3p negatively regulating the expression of HOXA10.

BIBR1532 Affects Endometrial Cell Proliferation, Migration, and Invasion in Endometriosis via Telomerase Inhibition and MAPK Signaling

Objectives: The effect of telomerase inhibitor BIBR1532 on endometriotic cells was investigated to explore the inhibitory effect of targeting telomerase on endometriosis. Design: In vitro primary cell culture study. Participants/Materials: Primary endometrial cells derived from eutopic and ectopic endometrium in patients with endometriosis. Setting: The study was conducted in the university hospital. Methods: Paired eutopic and ectopic endometrial cells were collected from 6 patients from January 2018 to July 2021. A TRAP assay was performed to detect the telomerase activity of the cells. MTT, cell cycle, apoptosis, migration, and invasion assays were performed to study the inhibitory effect of BIBR1532. Enrichment analysis was performed to identify the key pathways involved in endometriosis progression and telomerase action. Then, Western blotting was used to investigate the expression of related proteins. Results: BIBR1532 treatment significantly inhibited the growth of eutopic and ectopic endometrial cells, with apoptosis and cell cycle signaling involved. Migration and invasion, important characteristics for the establishment of ectopic lesions, were also inhibited by BIBR1532. The MAPK signaling cascade, related to telomerase and endometriosis, was decreased in eutopic and ectopic endometrial stromal cells with the treatment of BIBR1532. Limitations: The severe side effects of telomerase inhibitors might be the main obstacle to clinical application, so it is necessary to find better drug delivery methods in vivo. Conclusions: The telomerase inhibitor BIBR1532 affects endometrial cell proliferation, migration, and invasion in endometriosis.

WIF1 promoter hypermethylation induce endometrial carcinogenesis through the Wnt/β-catenin signaling pathway.

This study explores the mechanism underlying WIF1 promoter methylation and its relationship with the pathogenesis of endometrial carcinoma. WIF1 promoter methylation was detected using methylation-specific polymerase chain reaction (MSP). WIF1 expression was examined through qRT-PCR and western blotting. Furthermore, 5-aza-2'-deoxycytidine (5-Aza) was used to demethylate the WIF1 promoter. The roles of WIF1 were investigated using in vitro loss- and gain-of-function assays. Xenograft models were used to analyze WIF1 expression and downstream genes, and results were confirmed using immunofluorescence and western blotting. WIF1 promoter methylation in endometrial cancer cells was significantly higher than that in normal cells, but the WIF1 mRNA and protein levels were reduced. The expression of WIF1 increased significantly after 5-Aza treatment (p<.05). Thus, 5-Aza treatment can inhibit the proliferation of endometrial cancer cells and induce apoptosis, while knockdown of WIF1 significantly inhibits the effects of 5-Aza. 5-Aza treatment can also inhibit Wnt pathway genes, including phosphorylation of β-catenin protein, c-Myc, and CyclinD1, inhibit downstream functional genes, and activate the tumor suppressor gene APC, which can be blocked by WIF1 knockdown in endometrial carcinoma cells. Finally, 5-Aza inhibited the proliferation of subcutaneous tumor-bearing nude mice with endometrial cancer cells, but the effect was weaker than that of WIF1 overexpression. Our research shows that WIF1 promoter hypermethylation may promote the progression of endometrial cancer by downregulating WIF1 expression, activating the Wnt/β-catenin pathway, and promoting proliferation and inhibiting apoptosis. WIF1 may be a potential biological target for gene therapy and drug development for the treatment of endometrial cancer.

GRIK1-AS1 deficiency accelerates endometriosis progression by boosting DNMT1-dependent SFRP1 promoter methylation in endometrial stromal cells.

Endometriosis, a gynecological disease that affects up to 10% of women, is a major cause of pain and infertility. Deregulation of the epigenome is accountable for the onset and progression of endometriosis, although its exact mechanism is unknown. The purpose of the current study is to examine the role of the long non-coding RNA (lncRNA) GRIK1-AS1 in the epigenetic regulation of endometrial stromal cell proliferation and the development of endometriosis. Endometriosis datasets were screened to identify GRIKI-AS1 as dramatically declining in endometriosis. Gain or loss of function endometrial stromal cell (ESC) models were established. The anti-proliferation phenotype was investigated using in vitro and in vivo experiments. Epigenetic regulatory network analyses were conducted to suggest the intrinsic molecular mechanism. With bioinformatic and clinical data, we observed that GRIK1-AS1 and SFRP1 were expressed at low levels in endometriosis. Overexpressed GRIK1-AS1 inhibited ESC proliferation, while SFRP1 knockdown rescued the antiproliferative ability of GRIK1-AS1. Specifically, methylation-dependent expression inhibition of SFRP1 was revealed in ESCs. Mechanistically, GRIK1-AS1 hampers the occupancy of DNMT1 in SRFP1 promoter, leading to hypomethylation of SFRP1 and upregulated SFRP1 expression, thereby potentially suppressing Wnt signaling and its adverse proliferative effect. Therapeutically, lentivirus-mediated upregulation of GRIK1-AS1 inhibited endometriosis disease progression in vivo. Our study is a proof-of-concept demonstration for GRIKI-AS1-associated endometriosis pathogenesis and highlights a potential intervention target.

P-345 GM-CSF drives endometrial repair via p-STAT3 mediating angiogenesis

Abstract Study question Does granulocyte macrophage colony-stimulating factor (GM-CSF) play a role in endometrial angiogenesis and can it be used as a new application for treating endometrial regeneration? Summary answer GM-CSF improves endometrial repair via promoting endometrial angiogenesis and may provide a novel insight and therapeutics for endometrial injury. What is known already GM-CSF is a cytokine normally expressed in the female reproductive tract and plays key roles in embryo implantation and subsequent development. Our previous study found that intraperitoneally （i.p.）injection of GM-CSF can significantly improve endometrial repair by promoting endometrial glandular cells proliferation and stromal cells migration, increasing the thickness of endometrium and embryo implantation in mice. However, whether GM-CSF is involved in endometrial angiogenesis is unknown. Study design, size, duration To observe whether angiogenesis is promoted by GM-CSF, the expression of angiogenic factor CD31 was evaluated after injection of GM-CSF into endometrial injured mice model. The effect of GM-CSF on vascular g regeneration was also explored by zebrafish embryo model of intersegmental vascular injury. Human umbilical vein vascular endothelial cells (HUVECs) were cultured in vitro, and the role of GM-CSF on HUVECs were examined through EdU, Tube formation, Scratch repair and Transwell assays. Participants/materials, setting, methods 20μl 90% ethanol was used to establish endometrial injured mice model. After modeling, compared with i.p. injection of GM-CSF and saline, observing whether GM-CSF had the effect of repairing angiogenesis of injured endometrium, by evaluating expression of CD31. Real-time PCR, Western Blot were used to verify differentially expressed levels of mRNA and protein. Western Blot was used to confirm protein location. ChIP was used to analyze stat3 regulation of GM-CSF. Main results and the role of chance GM-CSF promoted expression of angiogenic factor CD31 in the mice model of endometrial injury. GM-CSF can repair and regenerate the zebrafish embryo model of intersegmental vascular injury caused by Sorafenib. GM-CSF can promote angiogenesis by HUVECs proliferation and migration, and significantly increase the formation of vascular-like network structures In GM-CSF treatment group, the mRNA expression of VEGF, MMP2, Ang1, Ang2 and Tie2 mRNA in HUVECs cells increased significantly; GM-CSF can activate the phosphorylation expression levels of p-FAK, p-Src, p-ERK 1/2, p-STAT3, p-p38 MAPK, pc-Jun, p-CREB, p-Akt, and p-eNOS. And it can increase the expression of downstream VEGF and MMP2 proteins. GM-CSF treatment for 30 min can promote phosphorylation and translocation of STAT3 from cytoplasm to nucleus, thereby regulating the expression of VEGF and MMP2 downstream. While FAK inhibitors (PF573228) and STAT3 knockdown can abrogate GM-CSF effect on HUVECs. Limitations, reasons for caution The results at the cellular and animal level can’t be completely mimic human endometrial injury. In future, well-designed clinical trials are needed to investigate the efficacy and safety of GM-CSF for treatment of human endometrial vascular injury. Wider implications of the findings GM-CSF can promote the vascular regeneration in mice model of endometrial injury through p-STAT3. Our findings provide new ideas for clinical treatment of endometrial injury. Trial registration number Not applicable

Melatonin modulates endometrial decidualization via NOTCH1-NRF2-FOXO1-GSH pathway†.

Melatonin is important for oocyte maturation, fertilization, early embryonic development, and embryo implantation, but less knowledge is available regarding its role in decidualization. The present study found that melatonin did not alter the proliferation of human endometrial stromal cells (ESCs), as well as cell cycle progress, but suppressed stromal differentiation after binding to the melatonin receptor 1B (MTNR1B), which was visualized in decidualizing ESCs. Further analysis evidenced that application of melatonin resulted in the diminishment for NOTCH1 and RBPJ expression. Supplementation of recombinant NOTCH1 protein (rNOTCH1) counteracted the impairment of stromal differentiation conferred by melatonin, while the addition of the NOTCH signaling pathway inhibitor DAPT aggravated the differentiation progress. Meanwhile, melatonin might restrain the expression and transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2), whose blockage accelerated the fault of stromal differentiation under the context of melatonin, but this restraint was subsequently ameliorated by rNOTCH1. Forkhead box O 1 (FOXO1) was identified as a downstream target of melatonin in decidualization. Repression of NRF2 antagonized the retrieval of rNOTCH1 due to aberrant FOXO1 expression elicited by melatonin. Moreover, melatonin brought about the occurrence of oxidative stress accompanied by an obvious accumulation of intracellular reactive oxygen species and a significant reduction in glutathione (GSH) content, as well as enzymatic activities of glutathione peroxidase and glutathione reductase, whereas supplementation of rNOTCH1 improved the above-mentioned effects. Nevertheless, this improvement was disrupted by the blockage of NRF2 and FOXO1. Furthermore, addition of GSH rescued the defect of stromal differentiation by melatonin. Collectively, melatonin might impair endometrial decidualization by restraining the differentiation of ESCs dependent on NOTCH1-NRF2-FOXO1-GSH pathway after binding to the MTNR1B receptor.

MenSCs Transplantation Improve the Viability of Injured Endometrial Cells Through Activating PI3K/Akt Pathway.

Endometrial injury is one of the leading causes of female infertility and is caused by intrauterine surgery, endometrial infection, repeated abortion, or genital tuberculosis. Currently, there is little effective treatment to restore the fertility of patients with severe intrauterine adhesions and thin endometrium. Recent studies have confirmed the promising therapeutic effects of mesenchymal stem cell transplantation on various diseases with definite tissue injury. The aim of this study is to investigate the improvements of menstrual blood-derived endometrial stem cells (MenSCs) transplantation on functional restoration in the endometrium of mouse model. Therefore, ethanol-induced endometrial injury mouse models were randomly divided into two groups: the PBS-treated group, and the MenSCs-treated group. As expected, the endometrial thickness and gland number in the endometrium of MenSCs-treated mice were significantly improved compared to those of PBS-treated mice (P < 0.05), and fibrosis levels were significantly reduced (P < 0.05). Subsequent results revealed that MenSCs treatment significantly promoted angiogenesis in the injured endometrium. Simultaneously, MenSCs enhance the proliferation and antiapoptotic capacity of endometrial cells, which is likely contributed by activating the PI3K/Akt signaling pathway. Further tests also confirmed the chemotaxis of GFP-labeled MenSCs towards the injured uterus. Consequently, MenSCs treatment significantly improved the pregnant mice and the number of embryos in pregnant mice. This study confirmed the superior improvements of MenSCs transplantation on the injured endometrium and uncovered the potential therapeutic mechanism, which provides a promising alternative for patients with serious endometrial injury.

Activation of SGK1/ENaC Signaling Pathway Improves the Level of Decidualization in Unexplained Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) is one of the most common complications during pregnancy and seriously affects women's physical and mental health. About 50% of RSA cases are of unknown etiology. Our previous study found that the decidual tissue of patients with unexplained recurrent spontaneous abortion (URSA) had low expression levels of serum and glucocorticoid-induced protein kinase (SGK) 1. Endometrial decidualization is a key link in the early stage of pregnancy and is crucial to the development and maintenance of pregnancy. Decidualization is the proliferation and differentiation of endometrial stromal cells into deciduals, which involves a complex physiological process such as ovarian steroid hormones (estrogen, progesterone, prolactin, etc.), growth factors, and intercellular signaling. The binding of estrogen and its receptor stimulates the synthesis of endometrial deciduating markers prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1), which mediates the occurrence of decidualization. Among them, SGK1/ENaC is a signaling pathway closely related to decidualization. The purpose of this study was to further investigate the expression of SGK1 and decidualization-related molecules in the decidual tissue of URSA patients and to explore the potential mechanism of SGK1's protective effect in URSA patients and in mouse models. Decidual tissue samples from 30 URSA patients and 30 women who actively terminated pregnancy were collected, and a URSA mouse model was established and treated with dydrogesterone. Expression levels of SGK1 and its signaling pathway-related proteins (p-Nedd4-2, 14-3-3 protein and ENaC-a), estrogen and progesterone receptors (ERβ, PR), and decidualization markers (PRLR, IGFBP-1) were assessed. Our study found that SGK1, p-Nedd4-2, 14-3-3 proteins, and ENaC-a expression levels were reduced in the decidual tissue, the SGK1/ENaC signaling pathway was inhibited, and the expression levels of the decidualization markers PRLR and IGFBP-1 were downregulated in the URSA group compared with the controls. Additionally, the concentrations of E2, P, and PRL in the serum of mice were decreased in the URSA group compared with the controls. However, SGK1/ENaC pathway-related proteins, estrogen and progesterone and their receptors, and decidualization-related molecules were upregulated by dydrogesterone. These data suggest that estrogen and progesterone can induce decidualization by activating the SGK1/ENaC signaling pathway; disruption of this pathway can lead to the development of URSA. Dydrogesterone can increase the expression level of SGK1 protein in decidual tissue.

Effect of Sparganii Rhizoma-Curcumae Rhizoma-medicated serum on proliferation, apoptosis, and migration of human ectopic endometrial stromal cells: based on JAK2/STAT3 signaling pathway

Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 μmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P&lt;0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P&lt;0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P&lt;0.05 or P&lt;0.01), down-regulate the expression of Bcl-2(P&lt;0.01), decrease the cell migration rate(P&lt;0.05 or P&lt;0.01), and reduce the secretion levels of IL-1β, IL-6, and TNF-α(P&lt;0.05 or P&lt;0.01), and levels of p-JAK2 and p-STAT3(P&lt;0.05 or P&lt;0.01). Compared with the SR and CR groups, CM group showed low cell viability(P&lt;0.01), high protein expression of caspase-3 and Bax(P&lt;0.05 or P&lt;0.01), and low protein expression of Bcl-2 and p-JAK2(P&lt;0.05). After incubation with CM, the apoptosis rate was higher(P&lt;0.05) and the migration rate was lower(P&lt;0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P&lt;0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.

Mesenchymal stem cell secretome alters gene expression and upregulates motility of human endometrial stromal cells.

Endometrial stromal cell motility is fundamental to regeneration and repair of this tissue and crucial for successful reproduction. This paper shows a role for the mesenchymal stem cell (MSC) secretome in enhancing endometrial stromal cell motility. Cyclic regeneration and repair of the endometrium are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, mechanisms remain unclear. This study tested the hypothesis that the BM-MSC and UC-MSC secretomes upregulate human endometrial stromal cell (HESC) proliferation, migration, and invasion and activate pathways to increase HESC motility. BM-MSCs were purchased from ATCC and cultured from the BM aspirate of three healthy female donors. UC-MSCs were cultured from umbilical cords of two healthy male term infants. Using indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system, we demonstrated that co-culture of HESCs with BM-MSCs or UC-MSCs from all donors significantly increased HESC migration and invasion, whereas effects on HESC proliferation varied among BM-MSC and UC-MSC donors. Analysis of gene expression by mRNA sequencing and RT-qPCR showed that expression of CCL2 and HGF was upregulated in HESCs that had been cocultured with BM-MSCs or UC-MSCs. Validation studies revealed that exposure to recombinant CCL2 for 48 h significantly increased HESC migration and invasion. Increased HESC motility by the BM-MSC and UC-MSC secretome appears to be mediated in part by upregulated HESC CCL2 expression. Our data support the potential for leveraging MSC secretome as a novel cell-free therapy to treat disorders of endometrial regeneration.

CircRNA3669 promotes goat endometrial epithelial cells proliferation via miR-26a/RCN2 to activate PI3K/AKT-mTOR and MAPK pathways

The development of receptive endometrium (RE) from pre-receptive endometrium (PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells (GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways, thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA (ceRNA) to upregulate Reticulocalbin-2 (RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol (E2) and progesterone (P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26a-RCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.

Development, characterization and evidence of anti-endometriotic activity of Phytocannabinoid-Rich nanoemulsions

During the last decades, the cannabinoid research for therapeutic purposes has been rapidly advancing, with an ever-growing body of evidence of beneficial effects for a wide sort of conditions, including those related to mucosal and epithelial homeostasis, inflammatory processes, immune responses, nociception, and modulating cell differentiation. β-caryophyllene (BCP) is a lipophilic volatile sesquiterpene, known as non-cannabis-derived phytocannabinoid, with documented anti-inflammatory, anti-proliferative and analgesic effects in both in vitro and in vivo models. Copaiba oil (COPA) is an oil-resin, mainly composed of BCP and other lipophilic and volatile components. COPA is reported to show several therapeutic effects, including anti-endometriotic properties and its use is widespread throughout the Amazonian folk medicine. COPA was nanoencapsulated into nanoemulsions (NE), then evaluated regarding the potential for transvaginal drug delivery and providing endometrial stromal cell proliferation in vitro. Transmission electron microscopy (TEM) showed that spherical NE were obtained with COPA concentration that varied from 5 to 7 wt%, while surfactant was maintained at 7.75 wt%. Dynamic light scattering (DLS) measurements showed droplet sizes of 30.03 ± 1.18, 35.47 ± 2.02, 43.98 ± 4.23 and PdI of 0.189, 0.175 and 0.182, respectively, with stability against coalescence and Ostwald ripening during 90 days. Physicochemical characterization results suggest that NE were able to both improve solubility and loading capacity, and increase thermal stability of COPA volatile components. Moreover, they showed slow and sustained release for up to eight hours, following the Higuchi kinetic model. Endometrial stromal cells from non-endometriotic lesions and ectopic endometrium were treated with different concentrations of COPA-loaded NE for 48 h to evaluate its effect on cell viability and morphology. The results suggested significant decrease in cell viability and morphological modifications in concentrations higher than 150 μg/ml of COPA-loaded NE, but not when cells were treated with the vehicle (without COPA). Given the relevance of Copaifera spp. species in folk medicine and their bio economical importance in the Amazon, the development of novel formulations to overcome the technological limitations related to BCP and COPA, is promising. Our results showed that COPA-loaded NE can lead to a novel, uterus-targeting, more effective and promising natural alternative treatment of endometriosis.

Gut dysbiosis-derived β-glucuronidase promotes the development of endometriosis

To explore the role of gut dysbiosis-derived β-glucuronidase (GUSB) in the development of endometriosis (EMs). 16S rRNA sequencing of stool samples from women with (n = 35) or without (n = 30) endometriosis and from a mouse model was conducted to assess gut microbiome changes and identify molecular factors influencing the development of endometriosis. Experiments invivo in an endometriosis C57BL6 mouse model and invitro verified the level of GUSB and its role in the development of EMs. Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-sen University; Guangdong Provincial Clinical Research Center for Obstetrical and Gynecological Diseases. Women of reproductive age with a histological diagnosis of endometriosis were enrolled in the endometriosis group (n = 35) and infertile or healthy age-matched women who had undergone a gynecological or radiological examination in the control group (n = 30). Fecal and blood samples were taken the day before surgery. Paraffin-embedded sections from 50 bowel endometriotic lesions, 50 uterosacral lesions, 50 samples without lesions, and 50 normal endometria were collected. None. Changes in the gut microbiome of patients with EMs and mice and the effect of β-glucuronidase on the proliferation and invasion of endometrial stromal cells and the development of endometriotic lesions were assessed. No difference in α and β diversity was found between patients with EMs and controls. Immunohistochemistry analysis showed higher β-glucuronidase expression in bowel lesions and uterosacral ligament lesions than in the normal endometrium (p<0.01). β-Glucuronidase promoted the proliferation and migration of endometrial stromal cells during cell counting kit-8, Transwell, and wound-healing assays. Macrophage levels, especially M2, were higher in bowel lesions and uterosacral ligament lesions than in controls, and β-glucuronidase promoted the M0 to M2 transition. Medium conditioned by β-glucuronidase-treated macrophages promoted endometrial stromal cell proliferation and migration. β-Glucuronidase increased the number and volume of endometriotic lesions and number of macrophages present in lesions in the mouse EMs model. This β-Glucuronidase promoted EMs development directly or indirectly by causing macrophage dysfunction. The characterization of the pathogenic role of β-glucuronidase in EMs has potential therapeutic implications.

Bisphenol S Increases Cell Number and Stimulates Migration of Endometrial Epithelial Cells.

To determine whether bisphenol S (BPS), a common substitute for bisphenol A (BPA), induces cell proliferation and migration in human endometrial epithelial cells (Ishikawa) and adult mouse uterine tissues. Human endometrial Ishikawa cells were exposed to low doses of BPS (1 nM and 100 nM) for 72 hours. Cell proliferation was assessed through the viability assays MTT and CellTiter-Glo®. Wound healing assays were also used to evaluate the migration potential of the cell line. The expression of genes related to proliferation and migration was also determined. Similarly, adult mice were exposed to BPS at a dose of 30 mg/kg body weight/day for 21 days, after which, the uterus was sent for histopathologic assessment. BPS increased cell number and stimulated migration in Ishikawa cells, in association with the upregulation of estrogen receptor beta (ESR2) and vimentin (VIM). In addition, mice exposed to BPS showed a significantly higher mean number of endometrial glands within the endometrium. Overall, in vitro and in vivo results obtained in this study showed that BPS could significantly promote endometrial epithelial cell proliferation and migration, a phenotype also observed with BPA exposure. Hence, the use of BPS in BPA-free products must be reassessed, as it may pose adverse reproductive health effects to humans.

Targeting YAP1 ameliorates progesterone resistance in endometriosis

Does YAP1 inhibition alleviate progesterone resistance in endometriosis? YAP1 inhibition reduces progesterone resistance in vitro and in vivo. Progesterone resistance not only causes treatment failure for endometriosis but also inhibits eutopic endometrial cell proliferation, dysregulates decidualization, and reduces the success rates of pregnancy. Hippo/yes-associated protein 1 (YAP1) signaling pathway plays an important role in the pathogenesis of endometriosis. Paraffin-embedded tissues containing paired endometriotic and endometrial specimens (n = 42) and serum samples isolated from normal controls (n = 15) or endometriotic patients with (n = 25) or without (n = 21) prior dienogest treatment were analyzed. A mouse model of endometriosis was also used to evaluate the effects of YAP1 inhibition on progesterone resistance. Primary endometriotic and endometrial stromal cells treated with YAP1 inhibitor or miR-21 mimic/inhibitor were used for the in vitro studies including decidualization induction, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation. Tissue specimens and serum from human and mouse were used for immunohistochemistry staining, exosome isolation, and microRNA (miRNA) quantification, respectively. Herein, we report, by using ChIP-PCR and RNA-IP, that YAP1 inhibits progesterone receptor (PGR) expression through upregulation of miR-21-5p. Upregulation of miR-21-5p not only reduces PGR expression but also inhibits endometrial stromal cell decidualization. Indeed, levels of YAP1 and miR-21-5p are inversely correlated with the level of PGR in human endometrial samples. In contrast, knockdown of YAP1 or treatment with verteporfin (VP), a YAP1 inhibitor, reduces miR-21-5p expression, thus leading to an increase in PGR expression in ectopic endometriotic stromal cells. In the mouse model of endometriosis, treatment with VP increases PGR expression and enhances decidualization. More importantly, VP synergistically increases the treatment effect of progestin in causing the regression of endometriotic lesions and improves the decidualization capability of the endometrium. Interestingly, treatment with dienogest, a synthetic progestin, reduces YAP1 and miR-21-5p expression in human cells and in the mouse model of endometriosis. Patients who received dienogest treatment for 6 months show a significant decrease in serum extracellular vesicle-associated miR-21-5p level. A public dataset (GSE51981) containing a large cohort of endometriotic tissues is available from the Gene Expression Omnibus (GEO). A large cohort of clinical samples is needed to verify the current diagnostic value of miR-21-5p in future studies. The reciprocal regulation of YAP1 and PGR suggests that combined YAP1 inhibitor and progestin may be a better therapeutic approach for treating endometriosis. This study was supported by the Ministry of Science and Technology, Taiwan (MOST-111-2636-B-006-012, MOST-111-2314-B-006-075-MY3, and MOST-106-2320-B-006-072-MY3). The authors have no conflict of interest to disclose.

Proteomic profiles and the function of RBP4 in endometrium during embryo implantation phases in pigs

BackgroundEndometrial receptivity plays a vital role in the success of embryo implantation. However, the temporal proteomic profile of porcine endometrium during embryo implantation is still unclear.ResultsIn this study, the abundance of proteins in endometrium on days 9, 10, 11, 12, 13, 14, 15 and 18 of pregnancy (D9, 10, 11, 12, 13, 14, 15 and 18) was profiled via iTRAQ technology. The results showed that 25, 55, 103, 91, 100, 120, 149 proteins were up-regulated, and 24, 70, 169, 159, 164, 161, 198 proteins were down-regulated in porcine endometrium on D10, 11, 12, 13, 14, 15 and 18 compared with that on D9, respectively. Among these differentially abundance proteins (DAPs), Multiple Reaction Monitoring (MRM) results indicated that S100A9, S100A12, HRG and IFI6 were differentially abundance in endometrial during embryo implantation period. Bioinformatics analysis showed that the proteins differentially expressed in the 7 comparisons were involved in important processes and pathways related to immunization, endometrial remodeling, which have a vital effect on embryonic implantation.ConclusionOur results reveal that retinol binding protein 4 (RBP4) could regulate the cell proliferation, migration and apoptosis of endometrial epithelial cells and endometrial stromal cells to affect embryo implantation. This research also provides resources for studies of proteins in endometrium during early pregnancy.

Effects of Growth Hormone on Adult Human Gonads: Action on Reproduction and Sexual Function.

Growth hormone (GH), which is commonly considered to be a promoter of growth and development, has direct and indirect effects on adult gonads that influence reproduction and sexual function of humans and nonhumans. GH receptors are expressed in adult gonads in some species including humans. For males, GH can improve the sensitivity of gonadotropins, contribute to testicular steroidogenesis, influence spermatogenesis possibly, and regulate erectile function. For females, GH can modulate ovarian steroidogenesis and ovarian angiogenesis, promote the development of ovarian cells, enhance the metabolism and proliferation of endometrial cells, and ameliorate female sexual function. Insulin-like growth factor-1 (IGF-1) is the main mediator of GH. In vivo, a number of the physiological effects of GH are mediated by GH-induced hepatic IGF-1 and local IGF-1. In this review, we highlight the roles of GH and IGF-1 in adult human gonads, clarify potential mechanisms, and explore the efficacy and the risk of GH supplementation in associated deficiency and assisted reproductive technologies. Besides, the effects of excess GH on adult human gonads are discussed as well.

Using Odontoblasts Derived from Dog Endometrial Stem Cells Encapsulated in Fibrin Gel Associated with BMP-2 in a Rat Pulp-Capping Model

This study aimed to treat dental injuries by utilizing one of the most advanced tissue engineering techniques. In this study, an in vitro model was employed to investigate the proliferation and odontogenic differentiation of canine endometrial stem cells (C-EnSCs). Furthermore, the dentin regeneration potential of odontoblast like-cells (OD) derived from C-EnSCs was assessed in rats. The C-EnSCs were isolated by the enzymatic method and identified by flow cytometry. The C-EnSCs were encapsulated in fibrin gel associated with signaling factors to create the proper conditions for cell growth and differentiation. Then, the OD cells were associated with bone morphologic protein-2 (BMP-2) to promote dentin formation in vivo. The animal model used to evaluate the regenerative effect of cells and biomaterials included the preparation of the left maxillary first molar of rats for direct pulp capping operation. Animals were divided into four groups: group 1, a control group without any treatment, group 2, which received fibrin, group 3, which received fibrin with ODs (fibrin/ODs), and group 4, which received fibrin with ODs and BMP-2 (fibrin/ODs/BMP-2). The morphological observations showed the differentiation of C-EnSCs into adipose, bone, neural cells, and ODs. Furthermore, the histomorphometric data of the treated teeth showed how fibrin gel and BMP2 at a concentration of 100 ng/mL provided an optimal microenvironment for regenerating dentin tissue in rats, which was increased significantly with the presence of OD cells within eight weeks. Our study showed that using OD cells derived from C-EnSCs encapsulated in fibrin gel associated with BMP2 can potentially be an appropriate candidate for direct pulp-capping and dentin regeneration.