Trophoblast Research Articles

Irisin Improves Preeclampsia by Promoting Embryo Implantation and Vascular Remodeling.

Preeclampsia is a pregnancy-specific disorder with unclear pathogenesis. Irisin, a recently identified exercise-induced factor, significantly influences lipid metabolism and cardiovascular function. Nonetheless, its role in trophoblast development during human placentation and the related intracellular signaling pathways remain poorly understood. We assessed peripheral blood irisin expression in early pregnancy among patients with preeclampsia and its correlation with key clinical indicators. In trophoblast cell lines and mice, we used exogenous irisin and viral knockdown to investigate functional changes. Phosphorylation-specific antibody arrays and dual-luciferase reporter assays were used to explore downstream molecular mechanisms, which were subsequently validated in trophoblast cell lines and relevant gene knockout mice. In early pregnancy, patients with preeclampsia exhibit decreased peripheral blood irisin levels, occurring earlier than traditional predictive markers, such as PLGF (placental growth factor) and sFlt-1 (soluble fms-like tyrosine kinase-1). Furthermore, irisin concentration is positively correlated with proteinuria and abnormal blood pressure during pregnancy. Exogenous irisin significantly enhanced trophoblast cell migration, invasion, and proliferation while inhibiting apoptosis. It also increased STAT (signal transducers and activators of transcription) 4 phosphorylation and its binding to the GLUT (glucose transporter)-3 promoter, resulting in elevated GLUT-3 expression and glucose uptake in trophoblast cells. In vivo, increased peripheral irisin promoted embryo implantation, vascular remodeling, and enhanced glucose uptake, whereas reduced irisin resulted in a preeclampsia-like phenotype characterized by elevated blood pressure, proteinuria, renal-placental dysfunction, adipose accumulation, and restricted fetal growth. Peripheral irisin improves preeclampsia by promoting embryo implantation and vascular remodeling through the activation of the STAT4/GLUT-3 pathway. Reduced peripheral irisin may contribute to preeclampsia-like pathologies. This study supports the advocacy for appropriate exercise during early pregnancy and provides new insights for preeclampsia prevention.

Immuno-PET/CT Imaging of Trop2 with [18F]AlF-RESCA-T4 Differentiates Lung Cancer from Inflammation.

Immuno-PET/CT imaging, a branch of molecular imaging, can noninvasively and specifically visualize biomarker expression across the body. Trophoblast cell surface antigen 2 (Trop2) is a pan-cancer biomarker and plays a crucial role in tumorigenesis through multiple signaling pathways. The study aims to develop and translate novel Trop2 single-domain antibody (sdAb) tracers for clinical use. Methods: Two sdAbs (i.e., His-tagged T4 and His-tag-free RT4) are recombinantly expressed in Chinese hamster ovary cells. The purities and binding kinetics are determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, high-performance liquid chromatography, and surface plasmon resonance assays. The AlF restrained complexing agent (RESCA) method is applied to develop 18F-labeled sdAb tracers ([18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4), followed by thorough preclinical imaging and blocking studies on tumor-bearing mice and a pilot clinical trial evaluating the clinical imaging safety and feasibility of [18F]AlF-RESCA-T4 immuno-PET/CT. Results: [18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4 possess high radiochemical purities. Preclinical imaging in the T3M-4 tumor model revealed prominent uptake (percentage injected dose/g) of [18F]AlF-RESCA-T4 (11.13 ± 1.53, n = 4) and [18F]AlF-RESCA-RT4 (8.83 ± 1.22, n = 4), which were significantly reduced by coinjection of unlabeled T4 and RT4 in blocking studies. The His-tag removal strategy further optimized the probe's invivo pharmacokinetics and reduced renal radioactivity accumulation without significantly decreasing tumor uptake. In a pilot clinical trial, [18F]AlF-RESCA-T4 immuno-PET/CT showed promising potency in annotating Trop2 expression and differentiating tumors from inflammatory diseases such as tuberculosis. Conclusion: [18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4 can specifically annotate Trop2 expression. Clinical [18F]AlF-RESCA-T4 immuno-PET/CT imaging can screen patients for Trop2-targeted therapies and differentiate lung inflammation from cancer.

Trop2-Targeted Molecular Imaging in Solid Tumors: Current Advances and Future Outlook.

Trophoblast cell surface antigen 2 (Trop2), a transmembrane glycoprotein, plays a dual role in physiological and pathological processes. In healthy tissues, Trop2 facilitates development and orchestrates intracellular calcium signaling. However, its overexpression in numerous solid tumors shifts its function toward driving cell proliferation and metastasis, thus leading to a poor prognosis. The clinical relevance of Trop2 is underscored by its utility as both a biomarker for diagnostic imaging and a target for therapy. Notably, the U.S. Food and Drug Administration (FDA) has approved sacituzumab govitecan (SG), a novel Trop2-targeted agent, for treating triple-negative breast cancer (TNBC) and refractory urothelial cancer, highlighting the significance of Trop2 in clinical oncology. Molecular imaging, a powerful tool for visualizing and quantifying biological phenomena at the molecular and cellular levels, has emerged as a critical technique for studying Trop2. This approach encompasses various modalities, including optical imaging, positron emission tomography (PET), single photon emission computed tomography (SPECT), and targeted antibodies labeled with radioactive isotopes. Incorporating Trop2-targeted molecular imaging into clinical practice is vital for the early detection, prognostic assessment, and treatment planning of a broad spectrum of solid tumors. Our review captures the latest progress in Trop2-targeted molecular imaging, focusing on both diagnostic and therapeutic applications across diverse tumor types, including lung, breast, gastric, pancreatic, prostate, and cervical cancers, as well as salivary gland carcinomas. We critically evaluate the current state by examining the relevant applications, diagnostic accuracy, therapeutic efficacy, and inherent limitations. Finally, we analyze the challenges impeding widespread clinical application and offer insights into strategies for advancing the field, thereby guiding future research endeavors.

Small and Long Non-Coding RNA Analysis for Human Trophoblast-Derived Extracellular Vesicles and Their Effect on the Transcriptome Profile of Human Neural Progenitor Cells

In mice, the fetal brain is dependent upon the placenta for factors that guide its early development. This linkage between the two organs has given rise to the term, the placenta–brain axis. A similar interrelationship between the two organs may exist in humans. We hypothesize that extracellular vesicles (EVs) released from placental trophoblast (TB) cells transport small RNA and other informational biomolecules from the placenta to the brain where their contents have pleiotropic effects. Here, EVs were isolated from the medium in which human trophoblasts (TBs) had been differentiated in vitro from induced pluripotent stem cells (iPSC) and from cultured iPSC themselves, and their small RNA content analyzed by bulk RNA-seq. EVs derived from human TB cells possess unique profiles of miRs, including hsa-miR-0149-3p, hsa-302a-5p, and many long non-coding RNAs (lncRNAs) relative to EVs isolated from parental iPSC. These miRs and their mRNA targets are enriched in neural tissue. Human neural progenitor cells (NPCs), generated from the same iPSC, were exposed to EVs from either TB or iPSC controls. Both sets of EVs were readily internalized. EVs from TB cells upregulate several transcripts in NPCs associated with forebrain formation and neurogenesis; those from control iPSC upregulated a transcriptional phenotype that resembled glial cells more closely than neurons. These results shed light on the possible workings of the placenta–brain axis. Understanding how the contents of small RNA within TB-derived EVs affect NPCs might yield new insights, possible biomarkers, and potential treatment strategies for neurobehavioral disorders that originate in utero, such as autism spectrum disorders (ASDs).

An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

We have previously reported that the calcineurin inhibitor macrolide immunosuppressant Tacrolimus (TAC, FK506) can promote the migration and invasion of the human-derived extravillous trophoblast cells conducive to preventing implantation failure in immune-complicated gestations manifesting recurrent implantation failure. Although the exact mode of action of TAC in promoting implantation has yet to be elucidated, the integral association of its binding protein FKBP12 with the inositol triphosphate receptor (IP3R) regulated intracellular calcium [Ca2+]i channels in the endoplasmic reticulum (ER), suggesting that TAC can mediate its action through ER release of [Ca2+]i. Using the immortalized human-derived first-trimester extravillous trophoblast cells HTR8/SVneo, our data indicated that TAC can increase [Ca2+]I, as measured by fluorescent live-cell imaging using Fluo-4. Concomitantly, the treatment of HTR8/SVneo with TAC resulted in a major dynamic reorganization in the actin cytoskeleton, favoring a predominant distribution of cortical F-actin networks in these trophoblasts. Notably, the findings that TAC was unable to recover [Ca2+]i in the presence of the IP3R inhibitor 2-APB indicate that this receptor may play a crucial role in the mechanism of action of TAC. Taken together, our results suggest that TAC has the potential to influence trophoblast migration through downstream [Ca2+]i-mediated intracellular events and mechanisms involved in trophoblast migration, such as F-actin redistribution. Further research into the mono-therapeutic use of TAC in promoting trophoblast growth and differentiation in clinical settings of assisted reproduction is warranted.

Low molecular weight heparins promote migration and invasion of trophoblast cells through regulating the PI3K/AKT signaling pathway.

Pregnant women confront a high risk of mortality due to preeclampsia (PE), which also results in severe challenges for newborns. Due to their efficient properties and minimal side effects, low molecular weight heparins (LMWHs) are extensively utilized by optimizing their molecular size. Nevertheless, there have been no reports regarding the alleviating effect of LMWHs on PE and the molecular mechanism underlying it. To examine the therapeutic impact of LMWHs on PE, we initially created a PE rat model and assessed the advantages of LMWHs on PE through Western blot, immunofluorescence, TUNEL, 24-h proteinuria determination, and other techniques. Furthermore, we examined the in vitro molecular mechanism of LMWHs therapy on PE using CCK-8, Transwell, Flow cytometry, Wound healing assay, and other techniques. LMWHs, when used in vivo, reduced the rise in blood pressure and 24-h proteinuria in rat models of PE. Additionally, they prevented trophoblast cell apoptosis in these rat models. In vitro, LMWHs demonstrated a significant ability to enhance the migration and invasion of HTR-8 and JEG-3 cells. Mechanistically, LMWHs mitigate the development of PE by activating the PI3K/AKT signaling pathway. According to our findings, the activation of the PI3K/AKT signaling pathway by LMWHs appears to provide relief for PE. Therefore, we have compelling evidence supporting the use of LMWHs as an efficient treatment for PE.

Novel application of metabolic imaging of early embryos using a light-sheet on-a-chip device: a proof-of-concept study.

Is it feasible to safely determine metabolic imaging signatures of nicotinamide adenine dinucleotide [NAD(P)H] associated auto-fluorescence in early embryos using a light-sheet on-a-chip approach? We developed an optofluidic device capable of obtaining high-resolution 3D images of the NAD(P)H autofluorescence of live mouse embryos using a light-sheet on-a-chip device as a proof-of-concept. Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. Indeed, in recent years, metabolic imaging has been investigated using highly advanced microscopy technologies such as fluorescence-lifetime imaging and hyperspectral microscopy. The potential safety of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67 h post metabolic imaging at the two-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system non-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). The microstructures were fabricated following a standard UV-photolithography process integrating light-sheet fluorescence microscopy into a microfluidic system, including on-chip micro-lenses to generate a light-sheet at the centre of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce two-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between the study groups. A convolutional neural network (ResNet 34) model using metabolic images was also trained. The optofluidic device was capable of obtaining high-resolution 3D images of live mouse embryos that can be linked to their metabolic activity. The system's design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet and fast imaging of the embryos (<2 s), while keeping a low dose of light exposure (16 J · cm-2 and 8 J · cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable of obtaining 30 times higher signal-noise-ratio images than images obtained with a confocal system (P < 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (P > 0.05; Yate's chi-squared test). Additionally, embryos with inhibited metabolic activity showed a decreased blastocyst formation rate of 22.2% compared to controls, as well as a 47% reduction in metabolic activity measured by metabolic imaging (P < 0.0001; t-test). This indicates that the optofluidic device was capable of producing metabolic images of live embryos by measuring NAD(P)H autofluorescence, allowing a novel and affordable approach. The obtained metabolic images of two-cell embryos predicted blastocyst formation with an AUC of 0.974. N/A. The study was conducted using a mouse model focused on early embryo development assessing illumination at the two-cell stage. Further safety studies are required to assess the safety and use of 405 nm light at the blastocyst stage by investigating any potential negative impact on live birth rates, offspring health, aneuploidy rates, mutational load, changes in gene expression, and/or effects on epigenome stability in newborns. This light-sheet on-a-chip approach is novel and after rigorous safety studies and a roadmap for technology development, potential future applications could be developed for ART. The overall cost-efficient fabrication of the device will facilitate scalability and integration into future devices if full-safety application is demonstrated. This work was partially supported by an Ideas Grant (no 2004126) from the National Health and Medical Research Council (NHMRC), by the Education Program in Reproduction and Development (EPRD), Department Obstetrics and Gynaecology, Monash University, and by the Department of Mechanical and Aerospace Engineering, Faculty of Engineering, Monash University. The authors E.V-O, R.N., V.J.C., A.N., and F.H. have applied for a patent on the topic of this technology (PCT/AU2023/051132). The remaining authors have nothing to disclose.

68Ga-MY6349 PET/CT imaging to assess Trop2 expression in multiple types of cancer.

Background Considering trophoblast cell surface antigen 2 (Trop2) is overexpressed in a wide range of human epithelial cancers, it presents an attractive target for the diagnosis and treatment of multiple types of cancer. Herein, we have developed a Trop2-specific radiotracer, 68Ga-MY6349, and present a prospective, investigator-initiated trial to explore the clinical values of 68Ga-MY6349 PET/CT. Methods In this translational study, 90 patients with 15 types of cancer, who underwent 68Ga-MY6349 PET/CT, were enrolled prospectively. Among them, 78 patients underwent paired 68Ga-MY6349 and 18F-FDG PET/CT, and 12 patients with prostate cancer underwent paired 68Ga-MY6349 and 68Ga-PSMA-11 PET/CT. Results Among the 90 patients across 15 types of cancer, 68Ga-MY6349 uptake in tumors was generally high but heterogeneous, varying among lesions, patients, and cancer types. Trop2 expression level determined by immunohistochemistry was highly correlated with 68Ga-MY6349 uptake at primary and metastatic tumor sites. 68Ga-MY6349 PET/CT showed higher tumor uptake (quantified by SUVmax) than 18F-FDG PET/CT in certain types of cancer, including breast (7.2 vs. 5.4, P < 0.001), prostate (9.2 vs. 3.0, P < 0.001), and thyroid cancers (8.5 vs. 3.7, P < 0.001). When compared with 68Ga-PSMA-11, 68Ga-MY6349 PET/CT exhibited comparable lesion uptake (12.2 vs. 12.5, P = 0.223) but a better tumor-to-background contrast (15.8 vs. 12.2, P < 0.001) for primary and metastatic prostate cancer, allowing visualization of more metastatic lesions. Conclusion 68Ga-MY6349 PET/CT is a non-invasive method for comprehensively assessing Trop2 expression in tumors, which can improve the diagnosis and staging for cancer patients, and aid in the decision-making for Trop2-targeted therapies and advancing personalized treatment.

Calcitriol prevents SARS-CoV spike-induced inflammation in human trophoblasts through downregulating ACE2 and TMPRSS2 expression

SARS-CoV-2, the causative virus of COVID-19, increases the risk of pregnancy complications including hypertensive disorders and placental inflammation. The spike glycoprotein mediates viral cell entry by interacting with the angiotensin-converting enzyme (ACE)2 in conjunction with the transmembrane serine protease 2 (TMPRSS2). ACE1, ACE2 and renin are components of the renin-angiotensin system (RAS), which regulates blood pressure. As the placenta expresses all these proteins, it is a target for SARS-CoV-2 and a source of blood pressure modulators. Noteworthy, an ACE1/ACE2 ratio imbalance can lead to RAS dysregulation and a bad prognosis in COVID-19 patients. Calcitriol, the most active vitamin D metabolite, negatively regulates RAS, reduces inflammation, and enhances antiviral immunity, thereby protecting against COVID-19 severity. However, contrasting information exists on the regulatory role of calcitriol upon RAS components and SARS-CoV-2 receptors; while the impact of calcitriol on spike-induced inflammation in placental cells has not been explored. Thus, we studied the effects of calcitriol on these parameters using the trophoblast cell line HTR-8/SVneo and primary syncytiotrophoblasts. By RT-qPCR, ELISA, and immunocytochemistry, we found that the spike enhanced proinflammatory cytokines expression and secretion, while calcitriol significantly downregulated this effect. Calcitriol also diminished ACE1, ACE2, TMPRSS2, and renin gene expression, as well as ACE1/ACE2 mRNA ratio. ConclusionsIn the human placenta, calcitriol reduced the gene expression of main RAS components and TMPRSS2, resulting in the inhibition of spike-induced inflammation. This outcome suggest that vitamin D participates in restricting SARS-CoV-2 placental infection by rendering trophoblasts less permissive to infection while helping to regulate maternal blood pressure and decreasing inflammation.

Circular RNA circ_0022707 impedes the progression of preeclampsia via the miR-3135b/GHR/PI3K/Akt axis.

Preeclampsia (PE) is a severe pregnancy complication linked to maternal and fetal health, yet its underlying causes and pathogenesis remain elusive. Circular RNA (circRNA), a form of non-coding RNA, is implicated in the progression of PE; nevertheless, the specific mechanism is not fully elucidated. This study aimed to identify and validate circRNAs that are pivotal in the pathophysiology of PE. Firstly, we constructed a ceRNA network using datasets from the GEO database and identified circ_0022707 as our study target. Then, using qRT-PCR analysis, we validated that circ_0022707 was downregulated in preeclamptic placentas compared to those of normal pregnant women. In situ hybridization assays revealed that circ_0022707 existed in placental villous trophoblast cells. Additionally, Pearson correlation analysis revealed a negative relationship between the expression of circ_0022707 and PE-related indicators (systolic and diastolic blood pressure, along with 24-h proteinuria levels). Furthermore, gain-of-function experiments confirmed that circ_0022707 could promote trophoblast cell proliferation and cell cycle progression while suppressing apoptosis. In vivo experiments using a preeclampsia-like mouse model also demonstrated that circ_0022707 administration could mitigate preeclampsia-like symptoms. Mechanistically, we confirmed that circ_0022707 functions through the miR-3135b/GHR/PI3K/Akt pathway in trophoblast cells. Overall, our study has provided insight into the important function of circ_002707 in the development of PE, enhancing our understanding of the disease's mechanism and proposing a viable therapeutic strategy for PE.

Progesterone peak influences embryonic developmental morphokinetics on trigger day? A retrospective study

ObjectivePremature Progesterone Rise (PPR) is characterized by elevated serum progesterone concentrations either towards the end of the follicular phase or on the trigger day, surpassing a pre-defined threshold value. Aim of the study is to evaluate the impact of PPR exceeding 1.5 ng/ml at the time of hCG-trigger on embryo morphokinetic parameters and to identify predictive biomarkers of in IntraCytoplasmic Sperm Injection (ICSI) cycles outcomes.MethodsIt is a retrospective study including patients underwent ICSI cycles in the period 2020–2023. 58 patients were recruited in the study group showing P levels in the trigger day greater than or equal to 1.5 ng/ml. A matching control group of 58 patients with P levels below 1.5 ng/ml was after selected. The general characteristics of these patients, including age, Body Mass Index (BMI), antral follicle count (AFC), anti-Müllerian hormone (AMH) and follicle-stimulating hormone (FSH) levels, the type of infertility and smoking/non-smoking patients, were recorded on the day of their initial visit. Subsequently, data were collected regarding the number of eggs retrieved, mature eggs, successfully fertilized eggs, and embryos reaching the blastocyst stage. Additionally, the timing of embryonic development and the quality of obtained blastocysts, as assessed by the degree of expansion and the characteristics of the inner cell mass (ICM) and trophectoderm (TE), were evaluated using Time-Lapse technology.ResultsElevated P levels exceeding 1.5 ng/ml on the trigger day were directly associated with a significantly larger number of antral follicles, consequently leading to a higher count of retrieved eggs, mature eggs, successfully fertilized eggs and embryos reaching the blastocyst stage. Furthermore, the analysis of morphokinetic parameters indicated faster division times and a notably greater number of high-grade blastocysts in the study group compared to the control group.ConclusionsP levels ≥ 1.5 ng/ml on the trigger day did not negatively impact embryonic morphokinetic parameters, instead resulting in faster embryo development in the initial stages.

BMP5 promotes trophoblast functions upon N-glycosylation via the BMP5-SMAD1/5 signaling pathway in preeclampsia

IntroductionPreeclampsia (PE) is one of the most common pregnancy-related complications worldwide and currently lacks an effective treatment. While trophoblast cell dysfunction has been identified as the fundamental cause of PE, the underlying mechanisms remain unclear. Bone morphogenetic protein 5 (BMP5) is a secreted glycoprotein highly expressed in the placenta that is involved in cell proliferation, migration, and invasion. However, the role and mechanism of BMP5 glycosylation of trophoblast cell function remain unclear. MethodsThe expression of BMP5 and N-glycosylation in preeclamptic placental tissues was investigated. We predicted and validated the N-glycosylation sites of BMP5. Additionally, we evaluated the effect of BMP5 N-glycosylation on the proliferation, migration, invasion, and angiogenesis of human immortalized trophoblastic HTR-8/SVneo cells. Furthermore, the role of N-glycosylated BMP5 in activating the BMP5-SMAD1/5 signaling pathway and regulating trophoblastic cell functions was explored. ResultsOur study reveals that PHA-E+L (recognizing branching N-glycans) reactive N-glycans and BMP5 expression levels are lower in preeclamptic villous tissues compared to normal placental tissues. Additionally, we demonstrated that BMP5 is an N-glycosylation-modified protein. Furthermore, N-glycosylated BMP5 promoted the functional trophoblastic cells (HTR-8/SVneo). We also revealed that N-glycosylation of BMP5 regulates multiple cell functions through the BMP5-SMAD1/5 signaling pathway. ConclusionN-glycosylated BMP5 promotes trophoblast cell proliferation, migration, invasion, and angiogenesis. This study provides mechanistic insight as to how N-glycosylation of BMP5 in trophoblast cells can contribute to the pathogenesis of preeclampsia and provides a new basis for its diagnosis and treatment.

Cross‐Species Insights into Trophoblast Invasion During Placentation Governed by Immune‐Featured Trophoblast Cells (Adv. Sci. 42/2024)

Cross‐Species Insights into Trophoblast Invasion During Placentation Governed by Immune‐Featured Trophoblast Cells (Adv. Sci. 42/2024)

Induction of FAM46C expression mediated by DNMT3A downregulation is involved in early-onset preeclampsia through gene body methylation

BackgroundAberrant methylation of genomic DNA has been found in preeclamptic placentas, which is characterized by elevated DNA methylation and hypermethylation of gene body regions, but the underlying mechanism is not yet fully understood. MethodsGlobal DNA methylation was assessed through ELISA and HPLC. The methylation sites were detected using the Illumina Human Methylation 450 K Microarray. The methylation level of FAM46C promoter and gene body was detected through the bisulfite sequencing. RNA-seq was utilized to investigate the mechanism by which DNMT3A and FAM46C mediate the migration and invasion of trophoblast cells. ResultsWe discovered that DNMT3A knockdown led to elevated levels of gene body methylation and FAM46C transcription. FAM46C downregulation completely rescued the suppressive effects caused by DNMT3A knockdown on the migration and invasion of trophoblast cells. Mechanistically, DNMT3A reduction led to an increase in the enrichment of DNMT3B and DNMT1 in the gene body region of FAM46C. The results of transcriptome sequencing showed that DNMT3A and FAM46C regulate the adhesion of trophoblast cells. Elevated expression of FAM46C and increased methylation levels within its gene body region were observed in extravillous trophoblast cells of early-onset preeclamptic placentas. ConclusionsDNMT3A-mediated aberrant FAM46C gene body methylation is relevant to the development of early-onset preeclampsia.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1%, fibroblasts: 33.1%, control: 27.3%). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(-). In Expt. 3, addition of 10% fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2%; 10% FCS, 43.4%; 1% ADSC lysate, 34.2%; 10% ADSC lysate, 48.1%). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10% addition, corresponding to 1 x 105 cells/mL, is as effective as 10% FCS without a decrease in cryotolerance due to the increased lipid content.

Evaluation of trophoblast cell surface antigen-2 (TROP2) protein expression in chemotherapy-resistant and metastatic breast carcinomas.

Evaluation of trophoblast cell surface antigen-2 (TROP2) protein expression in chemotherapy-resistant and metastatic breast carcinomas.

CDC42 deficiency leads to endometrial stromal cell senescence in recurrent implantation failure.

Does the downregulation of cell division cycle 42 (CDC42) protein in endometrial stroma lead to endometrial senescence in patients with recurrent implantation failure (RIF), and what is the potential mechanism? CDC42 deficiency causes endometrial stromal senescence and decidualization defects, impairing uterine receptivity of RIF patients, via activation of Wnt signaling pathway. Uterine aging is unique due to the cyclic remodeling and decidualization of endometrial tissue. Several transcriptomic studies have reported increased senescence in the endometrium in young patients with RIF. Our previous transcriptomic sequencing study discovered that endometrium from women with RIF showed downregulation of CDC42, which is an essential molecule affected by various senescence-related diseases. The endometrial samples of a total of 71 fertile control patients and 37 RIF patients were collected to verify the association between CDC42 expression and endometrial senescence of RIF patients. Primary endometrial stromal cells (EnSCs) were isolated from endometrial biopsies taken from patients without any endometrial complications and planning to undergo IVF, then subjected to adenovirus-mediated CDC42 knockdown and decidualization induction to explore the detailed mechanism by which CDC42 governs stromal senescence and decidualization. Wnt inhibitor XAV-939 was used to correct the endometrial senescence and decidualization defect. Senescence was determined by cell cycle arrest markers (e.g. P16, P21, and P53), SASP molecules (e.g. IL6 and CXCL8), and SA-β-gal staining. Masson's staining and Sirius Red staining were used to detect the endometrial fibrosis. Decidualization was evaluated by the mRNA expression and protein secretion of PRL and IGFBP1, F-actin immunostaining, and the BeWo spheroids 'in vitro implantation' model. Methods used to assess cell function included adenovirus transduction, RNA-sequencing, bioinformatic analysis, western blotting, RT-qPCR, ELISA, and immunofluorescence. Here, we observed remarkably increased levels of stromal senescence and fibrosis, along with stromal CDC42 deficiency, in the endometrium of patients with RIF (P < 0.001). Knockdown of CDC42 effectively induced premature senescence in EnSCs, leading to aberrant accumulation of senescent EnSCs and collagen deposition during decidualization. CDC42 deficiency in EnSCs restrained the decidualization differentiation and receptivity to trophoblast cells. Transcriptomic analysis revealed Wnt signaling activation as a critical downstream alteration in CDC42-deficient EnSCs. Mechanistically, CDC42 interacted with AKT competitively to impede the binding of GSK3β to AKT. Knockdown of CDC42 increased AKT-mediated phosphorylation of GSK3β to inactivate the Axin-GSK3β destruction complex, leading to accumulation and nuclear translocation of β-catenin. Importantly, Wnt signaling inhibitors partially corrected the endometrial senescence caused by CDC42 deficiency, and improved both decidualization and trophoblast invasion. RNA-seq data sets generated in this study have been deposited at the NCBI database with BioProject accession number PRJNA1102745. The present study was based on in vitro cell cultures. Further studies involving CDC42-regulated endometrial senescence are needed in knockout mice model and human endometrial assembloids. In addition to uncovering endometrial senescence in RIF, our findings underscore the significance of CDC42 in modulating EnSC senescence to maintain the decidualization function, and suggest Wnt signaling inhibitors as potential therapeutic agents for alleviating endometrial senescence. This work was supported by the National Natural Science Foundation of China [82271698 (R.J.), 82030040 (H.S.), 82288102 (H.W.), and 82371680 (G.Y.)]; the Natural Science Foundation of Jiangsu Province [BK20231117 (R.J.)]; and the Medical Science and Technology Development Foundation of Nanjing Department of Health [YKK23097 (Y.Z.)]. The authors declare no potential conflicts of interest.

IMPACT OF BLASTOCYST GRADES TO IMPLANTATION AND LIVE-BIRTH RATES IN WOMEN LESS THAN 30 YEARS WITH SINGLE EMBRYO TRANSFERS

Background:Morphological evaluation of blastocysts focusing on key factors such as expansion, inner cell mass (ICM) quality, and trophectoderm (TE) quality have been most widely used for embryo selection. The study emphasizes the importance of detailed blastocyst evaluation, highlighting that morphological variations can have substantial implications for ART outcomes. Methods:A total of 115women aged less than 30 years undergoing SET from from January 2023 to June 2023 were included in this study.The patients were divided into three groups based on their morphologic grading before embryo transfer: good-quality (n = 53), average-quality (n = 52) and poor-quality blastocysts (n = 10). The three morphologic groups pregnancy outcomes were examined, and the implantation rate was logistically regressed. Results:Good-quality blastocysts yielded a statistically significantly higher implantation rate (77%) than average / poor-quality blastocysts (44% and 20% respectively). Average-quality blastocysts still yielded in higher implantation rates (44%) compared to poor-quality blastocysts (20%). The baseline characteristics were analysed for significance using the Pearsons Chi-squared test. A P-value &lt; 0.05 was considered statistically significant. All the statistical analyses were performed using the social science statistics website with SPSS software. Conclusions:Higher blastocyst morphologic grading was associated with increased implantation rate for single embryo transfers. These findings suggest that evaluating blastocyst morphology is critical when selecting the best blastocyst.

Simulated ischaemia/reperfusion impairs trophoblast function through divergent oxidative stress- and MMP-9-dependent mechanisms.

Early-onset pre-eclampsia is believed to arise from defective placentation in the 1st trimester, leading to placental ischaemia/reperfusion (I/R) and oxidative stress. However, our current understanding of the effects of I/R and oxidative stress on trophoblast function is ambiguous in part due to studies exposing trophoblasts to hypoxia instead of I/R, and which report conflicting results. Here we present a model of simulated ischaemia/reperfusion (SI/R) to recapitulate the pathophysiological events of early-onset PE, by exposing 1st trimester cytotrophoblast HTR-8/SVneo cells to a simulated ischaemia buffer followed by reperfusion. We examined different ischaemia and reperfusion times and observed that 1h ischaemia and 24h reperfusion induced an increase in reactive oxygen species (ROS) production (p < 0.0001) and oxygen consumption rate (p < 0.01). SI/R-exposed trophoblast cells exhibited deficits in migration, proliferation and invasion (p < 0.01). While the deficits in migration and proliferation were rescued by antioxidants, suggesting a ROS-dependent mechanism, the loss of invasion was not affected by antioxidants, which suggests a divergent ROS-independent pathway. In line with this, we observed a decrease in MMP-9, the key regulatory enzyme necessary for trophoblast invasion (p < 0.01), which was similarly unaffected by antioxidants, and pharmacological inhibition of MMP-9 replicated the phenotype of deficient invasion (p < 0.01). Collectively, these data demonstrate that I/R impairs trophoblast migration and proliferation via a ROS-dependent mechanism, and invasion via a ROS-independent loss of MMP-9, disambiguating the role of oxidative stress and providing insights into the response of trophoblasts to I/R in the context of early-onset PE.

CircPCNXL2 promotes preeclampsia progression by suppressing trophoblast cell proliferation and invasion via miR-487a-3p/interferon regulatory factor 2 axis.

Preeclampsia (PE) has culminated in maternal and perinatal sickness and death across the world, affecting approximately 4.6% of pregnancies. Circular RNAs (circRNAs) have been linked to the biology of numerous pathologies, including PE. Here, we investigated the functional role of circPCNXL2 in the progression of PE. We employed the GEO database to get the expression profile of circPCNXL2 in patients with PE. This was followed by the detection of the expression of circPCNXL2 and miR-326 by qRT-PCR. The role of circPCNXL2 on trophoblast cell proliferation, migration, and invasion was confirmed with cell viability assays, the transwell assay, and the colony formation assay. Further, we employed dual luciferase, FISH, RNA pull-down assay and Western blot analysis to determine the interaction between the expression of circPCNXL2, miR-487a-3p, and IRF2. Findings from this study revealed that proliferation and migration of trophoblast cells were significantly increased in the HTR-8/SVneo cells after silencing circPCNXL2. Additionally, knockdown of circPCNXL2 remarkably increased miR-487a-3p expression, while IRF2 expression was remarkably reduced (P < 0.05), indicating the presence of complementary binding sequence on miR-487a-3p with which they sequester circPCNXL2. Rescue experiments revealed that interaction occurs between circPCNXL2, miR-487a-3p, and the IRF2 protein, indicating that circPCNXL2 expression elicits suppression of migration and proliferation of trophoblast cells via the miR-487a-3p/IRF2 pathway. We demonstrated that circPCNXL2 upregulation promotes pre-eclampsia by inhibiting proliferation and migration of trophoblast cells via the miR-487a-3p/IRF2 pathway or axis.