Role In Embryo Implantation Research Articles

ZEB1 Promotes Epithelial-Mesenchymal Transition of Endometrial Epithelial Cells and Plays a Critical Role in Embryo Implantation in Mice.

Zinc finger E-box binding homeobox 1 (ZEB1) promotes epithelial-mesenchymal transition (EMT) in carcinogenesis, but its role in embryo implantation has not yet been well studied. In the present study we evaluated the hypothesis that ZEB1-induced EMT is essential for embryo implantation in vivo. Endometrial epithelium from female Kunming mice (non-pregnant, and pregnant from day 2.5 to 6.5) were collected for assessment of mRNA/protein expression of ZEB1, and EMT markers E-cadherin and vimentin, by employment of real-time quantitative reverse transcription PCR, Western blot, and immunohistochemical staining. To test if knockdown of ZEB1 affects embryo implantation in vivo, mice received intrauterine injection of shZEB1 before the number of embryos implanted was counted. The results showed that, ZEB1 was highly expressed at both mRNA and protein levels in the mouse endometrium on day 4.5 of pregnancy, paralleled with down-regulated E-cadherin and up-regulated vimentin expression (P < 0.05). Intrauterine injection of shZEB1 markedly suppressed embryo implantation in mice (P < 0.01). Conclusively, the present work demonstrated that ZEB1 is essential for embryo implantation under in vivo condition, and is possibly due to its effect on modulation of endometrial receptivity through EMT.

The Roles of GDF-9, BMP-15, BMP-4 and EMMPRIN in Folliculogenesis and In Vitro Fertilization.

Growth differentiation factor 9 (GDF-9) contributes to early ovarian development and oocyte survival. Higher concentrations of GDF-9 in follicular fluid (FF) are associated with oocyte nuclear maturation and optimal embryo development. In in vitro fertilization (IVF), GDF-9 affects the ability of the oocyte to fertilize and subsequent embryonic development. Bone morphogenetic protein 15 (BMP-15) is involved in the regulation of ovarian function and affects oocyte development. During IVF, BMP-15 contributes to the formation of competent blastocysts. BMP-15 may play a role in embryo implantation by affecting endometrial receptivity. Bone morphogenetic protein 4 (BMP-4) is involved in the regulation of follicle growth and development and affects granulosa cell (GC) differentiation. In relation to IVF, BMP-4 is important for embryonic development, influences cell fate and differentiation, and plays a role in facilitating embryo-endometrial interactions during the implantation process. Extracellular matrix metalloproteinase inducer (EMMPRIN) is associated with ovulation and follicle rupture, promotes the release of mature eggs, and affects the modification of the extracellular matrix of the follicular environment. In IVF, EMMPRIN is involved in embryo implantation by modulating the adhesive properties of endometrial cells and promotes trophoblastic invasion, which is essential for pregnancy to occur. The purpose of the current article is to review the studies and recent findings of GDF-9, BMP-15, BMP-4 and EMMPRIN as fundamental factors in normal follicular development and in vitro fertilization.

LIF-STAT signaling in decidual cells: a possible role in embryo implantation and early pregnancy.

In this study, we investigate the effects of miRNA-138-5p and probable G-protein coupled receptor 124 (GPR124)-regulated inflammasome and downstream leukemia inhibitory factor (LIF)-STAT and adhesion molecule signaling in human decidual stromal cells. After informed consent was obtained from women aged 25-38 years undergoing surgical termination of the normal pregnancy and spontaneous miscarriage after 6-9 weeks of gestation, human decidual stromal cells were extracted from the decidual tissue. Extracellular vesicles (EVs) with microRNA (miRNA) between cells have been regarded as critical factors for embryo-maternal interactions on embryo implantation and programming of human pregnancy. MicroRNA-138-5p acts as the transcriptional regulator of GPR124 and the mediator of downstream inflammasome. LIF-regulated STAT activation and expression of integrins might influence embryo implantation. Hence, a better understanding of LIF-STAT and adhesion molecule signaling would elucidate the mechanism of microRNA-138-5p- and GPR124-regulated inflammasome activation on embryo implantation and pregnancy. Our results show that microRNA-138-5p, purified from the EVs of decidual stromal cells, inhibits the expression of GPR124 and the inflammasome, and activates the expression of LIF-STAT and adhesion molecules in human decidual stromal cells. Additionally, the knockdown of GPR124 and NLRP3 through siRNA increases the expression of LIF-STAT and adhesion molecules. The findings of this study help us gain a better understanding the role of EVs, microRNA-138-5p, GPR124, inflammasomes, LIF-STAT, and adhesion molecules in embryo implantation and programming of human pregnancy.

The Contribution of Proteomics in Understanding Endometrial Protein Expression in Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) poses a significant challenge in assisted reproductive technology (ART) outcomes. The endometrium plays a crucial role in embryo implantation, and its protein expression profile is integral in determining receptivity. Proteomics has emerged as a valuable tool in unraveling the molecular intricacies underlying endometrial receptivity and RIF. The aim of the present review is to analyze the contribution of proteomics to the understanding of endometrial protein expression in women with RIF, based on the results of significant proteomic studies. Medline/Pubmed databases were searched using keywords pertaining to proteomics combined with terms related to RIF. 15 studies were included in the present review. Several proteins have been found to exbibit differential expression in endometrial biopsies and fluid samples between fertile women and women with RIF during the receptive endometrial phase. The profile of endometrial proteins varied significantly among the studies. Nevertheless, similar changes in the expression levels of annexin-6, progesterone receptor, MMP-2, and MMP-9 in the endometrium of women with RIF, were found in more than one study indicating that certain proteins could potentially be effective biomarkers of endometrial receptivity. Proteomics contributes significantly to the understanding of protein expression in the endometrium of women with RIF and the analysis of proteins in endometrial fluid are promising for improving the clinical management of RIF.

B(a)P induces ovarian granulosa cell apoptosis via TRAF2-NFκB-Caspase1 axis during early pregnancy

Benzo(a)pyrene [B(a)P] is an environmental endocrine disruptor with reproductive toxicity. The corpus luteum (CL) of the ovary plays an important role in embryo implantation and pregnancy maintenance. Our previous studies have shown that B(a)P exposure affects embryo implantation and endometrial decidualization in mouse, but its effects and mechanisms on CL function remain unclear. In this study, we explore the mechanism of ovarian toxicity of B(a)P using a pregnant mouse model and an in vitro model of human ovarian granulosa cells (GCs) KGN. Pregnant mice were gavaged with corn oil or 0.2 mg/kg.bw B(a)P from pregnant day 1 (D1) to D7, while KGN cells were treated with DMSO, 1.0IU/mL hCG, or 1.0IU/mL hCG plus benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a B(a)P metabolite. Our findings revealed that B(a)P exposure damaged embryo implantation and reduced estrogen and progesterone levels in early pregnant mice. Additionally, in vitro, BPDE impaired luteinization in KGN cells. We observed that B(a)P/BPDE promoted oxidative stress (OS) and inflammation, leading to apoptosis rather than pyroptosis in ovaries and luteinized KGN cells. This apoptotic response was mediated by the activation of inflammatory Caspase1 through the cleavage of BID. Furthermore, B(a)P/BPDE inhibited TRAF2 expression and suppressed NFκB signaling pathway activation. The administration of VX-765 to inhibit the Caspase1 activation, over-expression of TRAF2 using TRAF2-pcDNA3.1 (+) plasmid, and BetA-induced activation of NFκB signaling pathway successfully alleviated BPDE-induced apoptosis and cellular dysfunction in luteinized KGN cells. These findings were further confirmed in the KGN cell treated with H2O2 and NAC. In conclusion, this study elucidated that B(a)P/BPDE induces apoptosis rather than pyroptosis in GCs via TRAF2-NFκB-Caspase1 during early pregnancy, and highlighting OS as the primary contributor to B(a)P/BPDE-induced ovarian toxicity. Our results unveil a novel role of TRAF2-NFκB-Caspase1 in B(a)P-induced apoptosis and broaden the understanding of mechanisms underlying unexplained luteal phase deficiency.

Association of Fibroblast Growth Factor-1 Promoter Polymorphism and its Serum Concentrations with Repeated Implantation Failure after In vitro Fertilisation: A Cross-sectional Study.

Fibroblast growth factors (FGFs) play a key role in embryo implantation and support endometrial trophoblastic interaction. The aim of the study was to evaluate the association between FGF-1 (rs34011) gene variety and its serum concentration with repeated implantation failure (RIF). The design of the study was a cross-sectional study. Four hundred infertile women with a history of RIF and 400 healthy women undergoing the first in vitro fertilisation-embryo transfer attempt with successful delivery (controls) were enrolled in the study. Genomic DNA was extracted from peripheral blood leucocytes and genotyped by Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction. Serum FGF-1 concentration was evaluated with enzyme-linked immunosorbent assay. The ANOVA test was used to analyse the difference between the means of the groups. In RIF group, the genotype frequencies of the GG, GA and AA were 59%, 33.5% and 7.5%, respectively, whereas in controls were 72.5%, 24% and 3.5%, respectively. The G and A allele frequencies in the RIF group were 75.75% and 24.25%, while in controls were 84.5% and 15.5%, respectively (P < 0.0001). We have also shown that serum FGF-1 concentration in RIF and control groups was 17 ± 3.55 and 23.62 ± 4.91 pg/mL, respectively (P = 0.008). We have also shown that AA genotype is significantly associated with decreased serum FGF-1 concentration in RIF (AA, GA and GG serum levels were 9.55 ± 2.65, 14 ± 3.35 and 22.55 ± 7.26 pg/mL, and in controls were 12.22 ± 2.27, 18.44 ± 5.98 and 26.66 ± 8.29 pg/mL, respectively). The current study suggests that a significant association between FGF-1 (rs34011) promoter polymorphism and its serum concentration with RIF. The study also suggests that AA genotype is linked to lower FGF-1 serum levels and may play a risk factor for RIF.

Ascertaining sensitive exposure biomarkers of various metal(loid)s to embryo implantation

Close relationships exist between metal(loid)s exposure and embryo implantation failure (EIF) from animal and epidemiological studies. However, there are still inconsistent results and lacking of sensitive metal(loid) exposure biomarkers associated with EIF risk. We aimed to ascertain sensitive metal(loid) biomarkers to EIF and provide potential biological explanations. Candidate metal(loid) biomarkers were measured in the female hair (FH), female serum (FS), and follicular fluid (FF) with various exposure time periods. An analytical framework was established by integrating epidemiological association results, comprehensive literature searching, and knowledge-based adverse outcome pathway (AOP) networks. The sensitive biomarkers of metal(loid)s along with potential biological pathways to EIF were identified in this framework. Among the concerned 272 candidates, 45 metal(loid)s biomarkers across six time periods and three biomatrix were initially identified by single-metal(loid) analyses. Two biomarkers with counterfactual results according to literature summary results were excluded, and a total of five biomarkers were further determined from 43 remained candidates in mixture models. Finally, four sensitive metal(loid) biomarkers were eventually assessed by overlapping AOP networks information, including Se and Co in FH, and Fe and Zn in FS. AOP networks also identified key GO pathways and proteins involved in regulation of oxygen species biosynthetic, cell proliferation, and inflammatory response. Partial dependence results revealed Fe in FS and Co in FH at their low levels might be potential sensitive exposure levels for EIF. Our study provided a typical framework to screen the crucial metal(loid) biomarkers and ascertain that Se and Co in FH, and Fe and Zn in FS played an important role in embryo implantation.

The role of the annexin A protein family at the maternal-fetal interface.

Successful pregnancy requires the tolerance of the maternal immune system for the semi-allogeneic embryo, as well as a synchrony between the receptive endometrium and the competent embryo. The annexin family belongs to calcium-regulated phospholipid-binding protein, which functions as a membrane skeleton to stabilize the lipid bilayer and participate in various biological processes in humans. There is an abundance of the annexin family at the maternal-fetal interface, and it exerts a crucial role in embryo implantation and the subsequent development of the placenta. Altered expression of the annexin family and dysfunction of annexin proteins or polymorphisms of the ANXA gene are involved in a range of pregnancy complications. In this review, we summarize the current knowledge of the annexin A protein family at the maternal-fetal interface and its association with female reproductive disorders, suggesting the use of ANXA as the potential therapeutic target in the clinical diagnosis and treatment of pregnancy complications.

Stem cell factor's role in enhancing the quality of fertilized and cloned porcine embryos for improved embryonic stem cell derivation.

Stem cell factor (SCF), a cytokine growth factor, is expressed in various tissues of the male and female reproductive organs, including the testis, ovary, and endometrium. Its primary function involves cell survival, differentiation, and proliferation, achieved through its binding to the c-kit receptor. This study aimed to scrutinize the effects of SCF treatment during in vitro culture (IVC) on both the developmental potential and the efficiency of establishing embryonic stem cells (ESCs) from fertilized and cloned porcine embryos. The rates of cleavage and blastocyst formation exhibited no significant differences between fertilized and cloned embryos, even with the addition of SCF. However, it's worth noting that embryos cloned with Cloud eGFP as donor cells demonstrated notably increased rates of hatched blastocysts when treated with SCF, and this increase was statistically significant (p < 0.05). Furthermore, following the complete dissection of the blastocysts, although there was no significant difference in the SCF-treated group, the area of expansion was significantly reduced (p < 0.01) in the group treated with the antagonistic blocker (ACK2) compared to both the control and SCF-treated groups. These outcomes suggest that the SCF/c-kit signaling pathway might play a pivotal role in embryo implantation. As anticipated, the efficiency of deriving ESCs was significantly higher (p < 0.01) in the group subjected to SCF treatment (12.82 ± 1.02%) compared to the control group (5.41 ± 2.25%). In conclusion, this study highlights the crucial role of SCF in enhancing the quality of porcine embryos, a vital step in obtaining high-quality ESCs.

Identification of m6A Modification Regulated by Dysregulated circRNAs in Decidua of Recurrent Pregnancy Loss.

N6-methyladenosine (m6A) modification is a prevalent modification of messenger ribonucleic acid (mRNA) in eukaryote cells and is closely associated with recurrent pregnancy loss (RPL). Circular RNAs (circRNAs) play critical roles in embryo implantation, trophoblast invasion and immune balance, which are important events during pregnancy. However, how m6A modification is regulated by circRNAs and the potential regulatory mechanism of circRNAs on RPL occurrence remain largely unclassified. We displayed the expression profiles of circRNAs and mRNAs in the decidua of normal pregnancies and RPL patients based on circRNA sequencing and the Gene Expression Omnibus database. A total of 936 differentially expressed circRNAs were identified, including 509 upregulated and 427 downregulated circRNAs. Differentially expressed circRNAs were enriched in immune, metabolism, signaling and other related pathways via the analysis of Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The competitive endogenous RNA (ceRNA) network was predicted to supply the possible role of circRNAs in RPL occurrence, and we further analyzed the profiles of nine m6A regulators (seven readers, one writer and one eraser) managed by circRNAs in this network. We also showed the expression profiles of circRNAs in the serum, trying to seek a potential biomarker to help in the diagnosis of RPL. These data imply that circRNAs are involved in pathogenesis of RPL by changing immune activities, metabolism and m6A modification in the ceRNA network. Our study might provide assistance in exploring the pathogenesis and diagnosis of RPL.

EGF-Enhanced GnRH-II Regulation in Decidual Stromal Cell Motility through Twist and N-Cadherin Signaling.

Crucial roles in embryo implantation and placentation in humans include the invasion of the maternal decidua by extravillous trophoblasts and the motile behavior of decidual endometrial stromal cells. The effects of the epidermal growth factor (EGF) and GnRH-II in the endometrium take part in early pregnancy. In the present study, we demonstrated the coaction of EGF- and GnRH-II-promoted motility of human decidual endometrial stromal cells, indicating the possible roles of EGF and GnRH-II in embryo implantation and early pregnancy. After obtaining informed consent, we obtained human decidual endometrial stromal cells from decidual tissues from normal pregnancies at 6 to 12 weeks of gestation in healthy women undergoing suction dilation and curettage. Cell motility was evaluated with invasion and migration assays. The mechanisms of EGF and GnRH-II were performed using real-time PCR and immunoblot analysis. The results showed that human decidual tissue and stromal cells expressed the EGF and GnRH-I receptors. GnRH-II-mediated cell motility was enhanced by EGF and was suppressed by the knockdown of the endogenous GnRH-I receptor and EGF receptor with siRNA, revealing that GnRH-II promoted the cell motility of human decidual endometrial stromal cells through the GnRH-I receptor and the activation of Twist and N-cadherin signaling. This new concept regarding the coaction of EGF- and GnRH-promoted cell motility suggests that EGF and GnRH-II potentially affect embryo implantation and the decidual programming of human pregnancy.

Friend leukemia integration 1 overexpression decreases endometrial receptivity and induces embryo implantation failure by promoting PART1 transcription in the endometrial epithelial cells.

In vitro fertilization-embryo transfer (IVF-ET) is a crucial assisted reproductive technology for treating infertility. However, recurrent implantation failure (RIF), a significant challenge in IVF-ET success, remains unresolved. This study aimed to explore the role and mechanism of FLI1 in endometrial receptivity and RIF. Differential endometrial cell proportions between patients with RIF and control subjects were assessed using single-cell RNA sequencing (scRNA-seq) analysis. The chromatin accessibility of FLI1 in the luteal endometrial tissue of patients with RIF and control subjects was examined using the single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). FLI1 mRNA and protein levels were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Cell viability and migration were examined via cell counting kit (CCK)-8 and scratch healing assays. Epithelial-mesenchymal transition markers were analyzed using western blotting. Mechanisms underlying FLI1's regulation of PART1 transcription and expression in endometrial epithelial cells were explored using chromatin immunoprecipitation and dual-luciferase reporter assays. Adeno-associated virus (AAV) carrying epithelial cell-specific FLI1/PART1 overexpression sequences was uterinely injected in mice to assess FLI1/PART1 effects. scRNA-seq revealed diminished endometrial epithelial cell proportions in RIF patients. Meanwhile, scATAC-seq indicated enhanced chromatin accessibility of FLI1 in these cells. FLI1 exhibited specific expression in RIF patients' endometrial epithelial cells. Specific FLI1 overexpression inhibited embryo implantation, while knockdown enhanced it. Pregnant mice injected with AAV encoding FLI1 overexpression had significantly lower implantation than AAV-negative controls. FLI1 binding to PART1 promoter heightened PART1 transcription and expression in endometrial epithelial cells. Rescue experiments illustrated FLI1's role in embryo implantation by boosting PART1 expression. PART1 was notably elevated in RIF patients' luteal endometrial tissue and non-receptive endometrial epithelial cells (HEC-1-A). Specific PART1 overexpression dampened embryo implantation, whereas knockdown promoted it. Pregnant mice injected with AAV encoding PART1 had lower implantation than negative controls. PART1 knockdown mitigated FLI1's inhibitory impact on HEC-1-A cell viability and migration. FLI1 overexpression in the endometrial epithelial cells of patients with RIF inhibited embryo implantation by binding to the PART1 promoter region to promote PART1 expression. These findings can aid in the development of novel therapeutic targets for RIF.

METTL3 regulatory TROAP can regulate the progression of non-small cell lung cancer through PI3K/AKT and EMT signaling pathway.

TROAP, interacts with trophinin and bystin, polys a key role in embryo implantation. TROAP is required for spindle assembly and centrosome integrity during the mitosis. TROAP has been described to promote tumorigenesis in a diverse range of cancer. We performed this study to assess the biological and clinical significance of TROAP in Non-small cell lung cancer. Forty-eight pairs of lung adenocarcinoma (LUAD) tissues and paraneoplastic tissues were collected. RT-qPCR, western bolt and immunohistochemistry assay was used to test TROAP RNA and protein expression not in LUAD tissues and paraneoplastic tissues but in LUAD cell lines and control cell lines. TROAP knockdown and overexpression vector were constructed and transfected into lung cancer cells. CCK-8, transwell, and wound healing assays were used to assess cell viability, migration, and invasion. The expression of PI3K/AKT and EMT signaling proteins and METTL3 were determined by western blot. We found the TROAP was enriched in NSCLC tissues and cell lines. TROAP knockdown inhibited cell proliferation, migration, invasion compared with control group in NSCLC. Mechanism analysis revealed that TROAP activated PI3K/AKT and EMT signaling pathway. To a certain extent, TROAP was regulated by METTL3. In a word, TROAP accelerated the progression of NSCLC through PI3K/AKT and EMT pathway, and TROAP might be considered as a novel target for NSCLC therapy.

Gene Expression Levels of CSF-1 and CSF-1R Endometrial under The Influence of Prolactin Level in Unexplained Miscarriage: A Case-Control Study.

Hormones such as prolactin, by influencing expression of the endometrial genes, play a pivotal role in embryo implantation and development. The present study aimed to evaluate serum level of prolactin and its effect on altering expression level of colony-stimulating factor-1 (CSF-1) and colony-stimulating factor-1 receptor (CSF-1R) genes in endometrial tissue during in vitro fertilization (IVF) pregnancy in the infertile women and recurrent pregnancy loss (RPL), compared to fertile women, who lost their pregnancies at gestational age <20 weeks. In this case-control study, 40 infertile women, 40 IVF pregnant women with RPL and 40 fertile women who lost their pregnancies at <20 weeks of gestation for unknown reasons were selected. Prolactin serum level was assessed using ELISA technique and expression of CSF-1 and CSF-1R genes was determined in endometrial tissue, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Mean prolactin level of the infertile group was 24.38 ± 1.43 ng/mL and it had statistically significant relationship with the fertile group (P<0.001). Expression level of the CSF-1 and CSF-1R genes were higher in the fertile than infertile groups by 2.88 times (P<0.0001) and 2.64 times (P<0.0001), while it was respectively 2.28 (P<0.0001) and 1.69 (P<0.0001) times higher compared to the RPL group. Risk factors for pregnancy loss, such as aging, increased body mass index (BMI), smoking and diabetes caused decreasing changes in gene expression (CSF-1 and CSF-1R ) and the differences were statistically significant, except in the infertile group. The present study showed a significant relationship of CSF-1 and CSF-1R expression levels with pregnancy loss. Risk factors such as aging, obesity, smoking and diabetes decreased both genes expression levels.

Multiomics approaches to uncover endometrial receptivity in embryo implantation: A mini-review

Successful implantation requires concerted interactions during the apposition, adhesion, and invasion of the embryo into a receptive endometrium. However, the embryo implantation rate for assisted reproduction remains low despite the transfer of good quality embryos. Changes in endometrial transcriptomics, proteomics, lipidomics, and even microbiota all play important roles in embryo implantation. Specifically, the expression of steroid hormone-regulated adhesive and anti-adhesive molecules during the embryo implantation window is becoming an area of increasingly intense research. This review a) summarizes the different molecules expressed in the receptive endometrium and b) proposes the use of surface protein markers to predict pregnancy outcomes from assisted reproduction.

P-480 An endometrial receptivity scoring system evaluated by ultrasonography in patients undergoing frozen-thawed embryo transfer: a prospective cohort study

Abstract Study question Can a scoring system based on ultrasound indicators evaluate the endometrial receptivity (ER) in a noninvasive and effective manner? Summary answer A noninvasive ultrasound scoring system was proposed, through which, a patient’s ER situation can be assessed in a noninvasive, efficient and accurate manner. What is known already Endometrial receptivity (ER) refers to the receptiveness of the endometrium to embryonic implantation and subsequent development. The endometrium plays a crucial role in embryo implantation, especially when the embryo quality is good. Ultrasound technology has become a routine method for ER evaluation. The independent evaluation value of various ultrasonic indicators is controversial. Some researchers designed a multi-index prediction system, but the prediction results vary. To further understand ER, we performed this prospective cohort study to help evaluate ER in a noninvasive and efficient manner. Study design, size, duration This prospective study included 197 infertile women undergoing FET from April 2019 to July 2021. Transvaginal three-dimensional ultrasound was performed on the transfer day to evaluate ER, including endometrial thickness, morphology, volume, movement, blood flow and flow index. Participants/materials, setting, methods This study was conducted at the Reproductive and Genetic Hospital of CITIC-Xiangya. The primary outcome of this study was the clinical pregnancy rate. All included patients were divided into a pregnant group and a nonpregnant group according to whether clinical pregnancy was achieved. Main results and the role of chance A total of 197 FET patients with 139 pregnancies (70.5%) were analysed. Primary infertility (adjusted odds ratio (aOR), 1.98; 95% confidence interval (CI), 1.01–3.882; P = 0.047) and more frequent endometrial peristalsis (aOR, 1.33; 95% CI, 1.028–1.722; P = 0.03) were protective factors against clinical pregnancy. Scores of 1 to 2 were given according to the relationship between different groups of ultrasonic indices and the clinical pregnancy rate (CPR). A patient’s ER score was the sum of the scores of the 6 items. The ER score was significantly higher in the pregnant group than that in the nonpregnant group (7.40 ± 1.73 versus 6.33 ± 1.99, P = 0.001). The CPR increased as the ER score increased; the CPR significantly improved for the group with total ER scores of &amp;lt; 6 to ≥ 6 (45.5% versus 75.6%, P = 0.001). Limitations, reasons for caution First, this study only included natural cycles of FET. The application for other populations remains to be verified. Second, perhaps with the progress of ultrasonic technology, there will be new and more direct indicators. Third, we did not further explore the relationship between ER and the live birth rate. Wider implications of the findings The scoring system provides a broader idea for guiding clinical practice to perform more efficient transplantation and provide a noninvasive evaluation of ER in the future. Trial registration number None

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

Aberrant activation of Notch1 signaling in the mouse uterine epithelium promotes hyper-proliferation by increasing estrogen sensitivity.

In mammals, the endometrium undergoes dynamic changes in response to estrogen and progesterone to prepare for blastocyst implantation. Two distinct types of endometrial epithelial cells, the luminal (LE) and glandular (GE) epithelial cells play different functional roles during this physiological process. Previously, we have reported that Notch signaling plays multiple roles in embryo implantation, decidualization, and postpartum repair. Here, using the uterine epithelial-specific Ltf-iCre, we showed that Notch1 signaling over-activation in the endometrial epithelium caused dysfunction of the epithelium during the estrous cycle, resulting in hyper-proliferation. During pregnancy, it further led to dysregulation of estrogen and progesterone signaling, resulting in infertility in these animals. Using 3D organoids, we showed that over-activation of Notch1 signaling increased the proliferative potential of both LE and GE cells and reduced the difference in transcription profiles between them, suggesting disrupted differentiation of the uterine epithelium. In addition, we demonstrated that both canonical and non-canonical Notch signaling contributed to the hyper-proliferation of GE cells, but only the non-canonical pathway was involved with estrogen sensitivity in the GE cells. These findings provided insights into the effects of Notch1 signaling on the proliferation, differentiation, and function of the uterine epithelium. This study demonstrated the important roles of Notch1 signaling in regulating hormone response and differentiation of endometrial epithelial cells and provides an opportunity for future studies in estrogen-dependent diseases, such as endometriosis.

Identification of miR-192 target genes in porcine endometrial epithelial cells based on miRNA pull-down.

MicroRNAs (miRNAs)-a class of small endogenous non-coding RNAs-are widely involved in post-transcriptional gene regulation of numerous physiological processes. High-throughput sequencing revealed that the miR-192 expression level appeared to be significantly higher in the blood exosomes of sows at early gestation than that in non-pregnant sows. Furthermore, miR-192 was hypothesized to have a regulatory role in embryo implantation; however, the target genes involved in exerting the regulatory function of miR-192 required further elucidation. In the present study, potential target genes of miR-192 in porcine endometrial epithelial cells (PEECs) were identified through biotin-labeled miRNA pull-down; functional and pathway enrichment analysis was performed via gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Bioinformatic analyses were concurrently used to predict the potential target genes associated with sow embryo implantation. In addition, double luciferase reporter vectors, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and Western blot were performed to verify the targeting and regulatory roles of the abovementioned target genes. A total of 1688 differentially expressed mRNAs were identified via miRNA pull-down. Through RT-qPCR, the accuracy of the sequencing data was verified. In the bioinformatics analysis, potential target genes of miR-192 appeared to form a dense inter-regulatory network and regulated multiple signaling pathways, such as metabolic pathways and the PI3K-Akt, MAPKs, and mTOR signaling pathways, that are relevant to the mammalian embryo implantation process. In addition, CSK (C-terminal Src kinase) and YY1 (Yin-Yang-1) were predicted to be potential candidates, and we validated that miR-192 directly targets and suppresses the expression of the CSK and YY1 genes. We screened 1688 potential target genes of miR-192 were screened, and CSK and YY1 were identified as miR-192 target genes. The outcomes of the present study provide novel insights into the regulatory mechanism of porcine embryo implantation and the identification of miRNA target genes.

Mechanism of human chorionic gonadotropin in endometrial receptivity via the miR‐126‐3p/PI3K/Akt/eNOS axis

Human chorionic gonadotropin (hCG) might affect endometrial receptivity, exerting integral roles in embryo implantation. This study explored the action of hCG in endometrial receptivity via the miR-126-3p/PIK3R2/PI3K/Akt/eNOS axis. The embryo implantation dysfunction (EID) mouse models were established by administrating mifepristone and human endometrial epithelial cells (EECs) were used for in vivo experiments, both followed by hCG treatment. Expression level of CD105 and protein levels of cadherin CD144 and CD146 in mice were determined by immunohistochemistry and Western blot. The levels of miR-126-3p and PIK3R2 mRNA and PIK3R2, p-PI3K p85 α, PI3K p110 α, p-Akt, Akt, p-eNOS, and eNOS protein levels were measured. Cell proliferation was evaluated by CCK-8 and EdU assays. The binding sites of miR-126-3p and PIK3R2 were predicted and verified. hCG-treated EECs were further transfected with miR-126-inhibitor for functional rescue experiments. hCG ameliorated endometrial receptivity in EID mice. Moreover, hCG promoted miR-126-3p and suppressed PIK3R2 in EID mice and EECs. miR-126-3p targeted PIK3R2. EEC proliferation was enhanced after hCG treatment but inhibited by miR-126-3p downregulation. Both in vivo and in vitro experiments validated that hCG activated the PI3K/Akt/eNOS pathway through the miR-126-3p/PIK3R2 axis. Collectively, hCG improves endometrial receptivity by activating the PI3K/Akt/eNOS pathway via regulating miR-126-3p/PIK3R2.