Fibronectin mediates activin A-promoted human trophoblast migration and acquisition of endothelial-like phenotype.

BACKGROUND: During human early placentation, a proportion of extravillous trophoblasts (EVTs) migrate to the maternal decidua, differentiating into endovascular EVTs to remodel spiral arteries and ensure the establishment of blood circulation at the maternal-fetal interface. Inadequate EVT migration and endovascular differentiation are closely associated with adverse pregnancy outcomes such as miscarriage. Activin A and fibronectin are both secretory molecules abundantly expressed at the maternal-fetal interface. Activin A has been reported to regulate EVT biological functions. However, whether fibronectin mediates activin A-promoted EVT migration and acquisition of endothelial-like phenotype as well as the underlying molecular mechanisms remain unknown. Additionally, the role of fibronectin in pregnancy establishment and maintenance warrants further investigation. METHODS: Primary and immortalized (HTR8/SVneo) human EVTs were used as in vitro study models. Cultured human first-trimester chorionic villous explants were utilized for ex vivo validation. A local fibronectin knockdown model in ICR mouse uteri, achieved by nonviral in vivo transfection with small interfering RNA (siRNA) targeting fibronectin 1 (si-Fn1), was employed to explore the roles of fibronectin in the establishment and maintenance of early pregnancy. RESULTS: Our results showed that activin A treatment significantly induced fibronectin 1 (FN1) mRNA expression and fibronectin protein production, which is essential for human trophoblast migration and endothelial-like tube formation. Both basal and activin A-upregulated fibronectin expression were abolished by the TGF-ß type I receptor inhibitor SB431542 or siRNA-mediated knockdown of activin receptor-like kinase (ALK4) or SMAD4. Moreover, activin A-increased trophoblast migration and endothelial-like tube formation were attenuated following the depletion of fibronectin. Fibronectin knockdown via intrauterine siRNA administration reduced CD31 and cytokeratin 8 (CK8) expression at the maternal-fetal interface, resulting in a decrease in the number of implantation sites and embryos. CONCLUSIONS: Our study demonstrates that activin A promotes trophoblast cell migration and acquisition of endothelial-like phenotype via ALK4-SMAD2/3-SMAD4-mediated fibronectin upregulation. Furthermore, through a local fibronectin knockdown model in mouse uteri, we found that the absence of fibronectin at the maternal-fetal interface impedes endovascular migration of trophoblasts and decidual vascularization, thereby interfering with early embryo implantation and the maintenance of pregnancy. These findings provide novel insights into placental development during early pregnancy establishment and contribute to the advancement of therapeutic approaches for managing pregnancy complications related to trophoblast dysfunction.

Endometrial BMP2 Deficiency Impairs ITGB3-Mediated Trophoblast Invasion in Women With Repeated Implantation Failure.

BACKGROUND: Repeated implantation failure (RIF) leads to a waste of high-quality embryos and remains a challenge in assisted reproductive technology. During early human placentation, the invasion of trophoblast cells into the decidua is an essential step for the establishment of maternal-fetal interactions and subsequent successful pregnancy. Bone morphogenetic protein 2 (BMP2) has been reported to regulate endometrial receptivity and promote trophoblast invasion. However, whether there is dysregulation of endometrial BMP2 expression in patients with RIF remains unknown. Additionally, the molecular mechanisms underlying the effects of BMP2 on human trophoblast invasion and early placentation remain to be further elucidated. METHODS: Midluteal phase endometrial samples were biopsied from patients with RIF and from routine control in vitro fertilization followed by quantitative polymerase chain reaction and immunoblotting analyses. Human trophoblast organoids, primary human trophoblast cells, and an immortalized trophoblast cell line (HTR8/SVneo) were used as study models. RESULTS: We found that BMP2 was aberrantly low in midluteal phase endometrial tissues from patients with RIF. Recombinant human BMP2 treatment upregulated integrin ß3 (ITGB3) in a SMAD2/3-SMAD4 signaling-dependent manner in both HTR8/SVneo cells and primary trophoblast cells. siRNA-mediated integrin ß3 downregulation reduced both basal and BMP2-upregulated trophoblast invasion and vascular mimicry in HTR8/SVneo cells. Importantly, shRNA-mediated ITGB3 knockdown significantly decreased the formation ability of human trophoblast organoids. CONCLUSION: Our results demonstrate endometrial BMP2 deficiency in patients with RIF. ITGB3 mediates both basal and BMP2-promoted human trophoblast invasion and is essential for early placentation. These findings broaden our knowledge regarding the regulation of early placentation and provide candidate diagnostic and therapeutic targets for RIF clinical management.

CD8+ T cells reduce neuroretina inflammation in mouse by regulating autoreactive Th1 and Th17 cells through IFN-γ.

Various regulatory CD8+ T-cell subsets have been proposed for immune tolerance and have been implicated in controlling autoimmune diseases. However, their phenotypic identities and suppression mechanisms are not yet understood. This study found that coculture of T-cell receptor (TCR)- or interferon (IFN)-ß-activated CD8+ T cells significantly suppressed the cytokine production of Th1 and Th17 cells. By experimenting with the experimental autoimmune uveitis (EAU), we found that adoptive transfer of TCR or IFN-ß-activated CD8+ T cells significantly lessened disease development in an IFN-γ-dependent manner with a decreased uveitogenic Th1 and Th17 response. Interestingly, after adoptive transfer into the EAU mice, the IFN-γ+ CD8+ T cells were recruited more efficiently into the secondary lymphoid organs during the disease-priming phase. This recruitment depends on the IFN-γ-inducible chemokine receptor CXCR3; knocking out CXCR3 abolishes the protective effect of CD8+ T cells in EAU. In conclusion, we identified the critical role of IFN-γ for CD8+ T cells to inhibit Th1 and Th17 responses and ameliorate EAU. CXCR3 is necessary to recruit IFN-γ+ CD8+ T cells to the secondary lymphoid organ for the regulation of autoreactive Th1 and Th17 cells.

H3K27me3-modulated Hofbauer cell BMP2 signalling enhancement compensates for shallow trophoblast invasion in preeclampsia.

BACKGROUND: Preeclampsia (PE) is a common hypertensive pregnancy disorder associated with shallow trophoblast invasion. Although bone morphogenetic protein 2 (BMP2) has been shown to promote trophoblast invasion in vitro, its cellular origin and molecular regulation in placenta, as well as its potential role in PE, has yet to be established. Additionally, whether BMP2 and/or its downstream molecules could serve as potential diagnostic or therapeutic targets for PE has not been explored. METHODS: Placentas and sera from PE and healthy pregnant women were subjected to multi-omics analyses, immunoblots, qPCR, and ELISA assays. Immortalized trophoblast cells, primary cultures of human trophoblasts, and first-trimester villous explants were used for in vitro experiments. Adenovirus expressing sFlt-1 (Ad Flt1)-induced PE rat model was used for in vivo studies. FINDINGS: We find globally decreased H3K27me3 modifications and increased BMP2 signalling in preeclamptic placentas, which is negatively correlated with clinical manifestations. BMP2 is derived from Hofbauer cells and epigenetically regulated by H3K27me3 modification. BMP2 promotes trophoblast invasion and vascular mimicry by upregulating BMP6 via BMPR1A-SMAD2/3-SMAD4 signalling. BMP2 supplementation alleviates high blood pressure and fetal growth restriction phenotypes in Ad Flt1-induced rat PE model. INTERPRETATION: Our findings demonstrate that epigenetically regulated Hofbauer cell-derived BMP2 signalling enhancement in late gestation could serve as a compensatory response for shallow trophoblast invasion in PE, suggesting opportunities for diagnostic marker and therapeutic target applications in PE clinical management. FUNDING: National Key Research and Development Program of China (2022YFC2702400), National Natural Science Foundation of China (82101784, 82171648, 31988101), and Natural Science Foundation of Shandong Province (ZR2020QH051, ZR2020MH039).

Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage.

STUDY QUESTION: Can whole exome sequencing (WES) followed by trio bioinformatics analysis identify novel pathogenic genetic causes of first trimester euploid miscarriage? SUMMARY ANSWER: We identified genetic variants in six candidate genes that indicated plausible underlying causes of first-trimester euploid miscarriage. WHAT IS KNOWN ALREADY: Previous studies have identified several monogenic causes of Mendelian inheritance in euploid miscarriages. However, most of these studies are without trio analyses and lack cellular and animal models to validate the functional effect of putative pathogenic variants. STUDY DESIGN, SIZE, DURATION: Eight unexplained recurrent miscarriage (URM) couples and corresponding euploid miscarriages were included in our study for whole genome sequencing (WGS) and WES followed by trio bioinformatics analysis. Knock-in mice with Rry2 and Plxnb2 variants and immortalized human trophoblasts were utilized for functional study. Additional 113 unexplained miscarriages were included to identify the mutation prevalence of specific genes by multiplex PCR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole blood from URM couples and their <13 weeks gestation miscarriage products were both collected for WES, and all variants in selected genes were verified by Sanger sequencing. Different stage C57BL/6J wild-type mouse embryos were collected for immunofluorescence. Ryr2N1552S/+, Ryr2R137W/+, Plxnb2D1577E/+, and Plxnb2R465Q/+ point mutation mice were generated and backcrossed. Matrigel-coated transwell invasion assays and wound-healing assays were performed using HTR-8/SVneo cells transfected with PLXNB2 small-interfering RNA and negative control. Multiplex PCR was performed focusing on RYR2 and PLXNB2. MAIN RESULTS AND THE ROLE OF CHANCE: Six novel candidate genes, including ATP2A2, NAP1L1, RYR2, NRK, PLXNB2, and SSPO, were identified. Immunofluorescence staining showed that ATP2A2, NAP1L1, RyR2, and PLXNB2 were widely expressed from the zygote to the blastocyst stage in mouse embryos. Although compound heterozygous mice with Rry2 and Plxnb2 variants did not show embryonic lethality, the number of pups per litter was significantly reduced when backcrossing Ryr2N1552S/+ ♂ with Ryr2R137W/+ ♀ or Plxnb2D1577E/+ ♂ with Plxnb2R465Q/+ ♀ (P < 0.05), which were in accordance with the sequencing results of Family 2 and Family 3, and the proportion of Ryr2N1552S/+ offspring was significantly lower when Ryr2N1552S/+ female mice were backcrossed with Ryr2R137W/+ male mice (P < 0.05). Moreover, siRNA-mediated PLXNB2 knockdown inhibited the migratory and invasive abilities of immortalized human trophoblasts. Besides, additional 10 variants of RYR2 and PLXNB2 were detected in 113 unexplained euploid miscarriages by multiplex PCR. LIMITATIONS, REASONS FOR CAUTION: The relatively small number of samples is a limitation of our study which may result in the identification of variants in unique candidate genes with no definitive although plausible causal effect. Larger cohorts are needed to replicate these findings and additional functional research is needed to confirm the pathogenic effects of these variants. Moreover, the sequencing coverage restricted the detection of low-level parental mosaic variants. WIDER IMPLICATIONS OF THE FINDINGS: For first-trimester euploid miscarriage, variants in unique genes may be underlying genetic etiologies and WES on trio could be an ideal model to identify potential genetic causes, which could facilitate individualized precise diagnostic and therapeutic regimens in the future. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700604), National Natural Science Foundation of China (31900492, 82101784, 82171648), Basic Science Center Program of the National Natural Science Foundation of China (31988101), Key Research and Development Program of Shandong Province (2021LCZX02), Natural Science Foundation of Shandong Province (ZR2020QH051), Natural Science Foundation of Jiangsu Province (BK20200223), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201812154) and Young Scholars Program of Shandong University. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Multifunctional Interleukin-24 Resolves Neuroretina Autoimmunity via Diverse Mechanisms.

IL-24 is a multifunctional cytokine that regulates both immune cells and epithelial cells. Although its elevation is associated with a number of autoimmune diseases, its tolerogenic properties against autoreactive T cells have recently been revealed in an animal model of central nervous system (CNS) autoimmunity by inhibiting the pathogenic Th17 response. To explore the potential of IL-24 as a therapeutic agent in CNS autoimmunity, we induced experimental autoimmune uveitis (EAU) in wildtype mice and intravitreally injected IL-24 into the inflamed eye after disease onset. We found that the progression of ocular inflammation was significantly inhibited in the IL-24-treated eye when compared to the control eye. More importantly, IL-24 treatment suppressed cytokine production from ocular-infiltrating, pathogenic Th1 and Th17 cells. In vitro experiments confirmed that IL-24 suppressed both Th1 and Th17 differentiation by regulating their master transcription factors T-bet and RORγt, respectively. In addition, we found that intravitreal injection of IL-24 suppressed the production of proinflammatory cytokines and chemokines from the retinas of the EAU-inflamed eyes. This observation appears to be applicable in humans, as IL-24 similarly inhibits human retinal pigment epithelium cells ARPE-19. In conclusion, we report here that IL-24, as a multifunctional cytokine, is capable of resolving ocular inflammation in EAU mice by targeting both uveitogenic T cells and RPE cells. This study sheds new light on IL-24 as a potential therapeutic candidate for autoimmune uveitis.

Imaging-Based Body Fat Distribution in Polycystic Ovary Syndrome: A Systematic Review and Meta-Analysis.

Background: Women with polycystic ovary syndrome (PCOS) are generally considered to be central obese and at higher risks of metabolic disturbances. Imaging methods are the golden standards for detecting body fat distribution. However, evidence based on magnetic resonance imaging (MRI) and computed tomography (CT) is conflicting. This study systematically reviewed the imaging-based body fat distribution in PCOS patients and quantitatively evaluated the difference in body fat distribution between PCOS and BMI-matched controls. Methods: PUBMED, EMBASE, and Web of Science were searched up to December 2019, and studies quantitatively compared body fat distribution by MRI, CT, ultrasound, or X-ray absorptiometry (DXA) between women with PCOS and their BMI-matched controls were included. Two researchers independently reviewed the articles, extract data and evaluated the study quality based on Newcastle-Ottawa Scale (NOS). Results: 47 studies were included in systematic review and 39 were eligible for meta-analysis. Compared to BMI-matched controls, higher accumulations of visceral fat (SMD 0.41; 95%CI: 0.23-0.59), abdominal subcutaneous fat (SMD 0.31; 95%CI: 0.20-0.41), total body fat (SMD 0.19; 95% CI: 0.06-0.32), trunk fat (SMD 0.47; 95% CI: 0.17-0.77), and android fat (SMD 0. 36; 95% CI: 0.06-0.66) were identified in PCOS group. However, no significant difference was identified in all the above outcomes in subgroups only including studies using golden standards MRI or CT to evaluate body fat distribution (SMD 0.19; 95%CI: -0.04-0.41 for visceral fat; SMD 0.15; 95%CI: -0.01-0.31 for abdominal subcutaneous fat). Moreover, meta-regression and subgroup analyses showed that young and non-obese patients were more likely to accumulate android fat. Conclusions: PCOS women seem to have abdominal fat accumulation when compared with BMI-matched controls. However, MRI- and CT- assessed fat distribution was similar between PCOS and controls, suggesting central obesity may be independent of PCOS. These findings will help us reappraise the relationship between PCOS and abnormal fat deposition and develop specialized lifestyle interventions for PCOS patients. Systematic Review Registration: PROSPERO, identifier CRD42018102983.

[Determination of fifteen beta-agonists in animal urine by high performance liquid chromatography-tandem mass spectrometry].

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) method was established for the determination of fifteen beta-agonists (clenbuterol, ractopamine, salbutamol, cimaterol, mabuterol, tulobuterol, bambuterol, mapenterol, cimbuterol, zilpaterol, formoterol, clorprenaline, terbutaline, penbutolol and brombuterol) in animal urine. Perchloric acid solution was used to acidify the sample and precipitate protein in the sample. The sample was purified and concentrated by an HLB mini-column. The separation of the beta-agonist was performed on an Agilent 1100 HPLC system with a Eclipse XDB-C18 column by using gradient elution with methanol and water (containing 0.1% (v/v) formic acid) as the mobile phases at a flow rate of 1 mL/min. Qualitative and quantitative analysis of the fifteen beta-agonists, which were ionized by electrospray ionization interface (ESI), were carried out in multiple reaction monitoring (MRM) mode with API 4000 tandem mass spectrometry. The calibration curves showed good linearity in the mass concentration range of 0.25 - 20 microg/L with the correlation coefficients r > or = 0.999 5. The recoveries of the fifteen beta-agonists ranged from 62.1% to 107% at the spiked levels of 0.25, 1.0 and 10 microg/L. The relative standard deviations (n = 10) were between 3.5% and 9.9%. The limits of quantification (S/N > 10) were 0.25 microg/L for all the analytes. This method is simple, rapid, sensitive and accurate.