New data indicate an increased risk of obstetric complications after Covid-19, the aim of this study was therefore to investigate the expression of immune checkpoint molecules TIM-3 and Gal-9 in tissue sections of placenta from patients with acute Covid-19, from patients after Covid-19 infection and from healthy controls. We used a total of 60 placentas from women who had given birth to female or male children at Augsburg University Hospital, Germany. We examined 10 placentas each with acute Covid-19 from female and male fetuses, 10 placentas each from female and male fetuses after acute Covid-19 and 10 placentas each from female and male fetuses of a healthy control group. Immunohistochemical staining against TIM-3 and Gal-9 was performed, and marker expression was assessed by an immunoreactive score (IRS). The identity of cells expressing TIM-3 or Gal-9 was then analyzed by double immunofluorescence analysis. TIM-3- and Gal-9 positive macrophages are significantly upregulated in the decidua of female acute Covid-19 placentas. TIM-3 is significantly up-regulated in male extravillous trophoblast cells in the acute phase of Covid-19. On the other hand, TIM-3 was significantly downregulated in Hofbauer cells from male placentas after Covid-19. Gal-9, the ligand for TIM-3, was significantly downregulated in Hofbauer cells from male placentas after Covid-19. Triple immunofluorescence analysis showed that male acute Covid-19 Hofbauer cells (analyzed by CD163 staining) were positive for both TIM-3 and Gal-9. Surprisingly, the observed effects were related to fetal sex. In particular, the formative effect of this infection needs to be analyzed in further studies.

( Acta Obstet Gynecol Scand . 2025;104:151- 163. doi: 10.1111/aogs.15020) This study investigates a novel diagnostic approach utilizing a Multiple Marker Microarray Analyzer to detect preeclampsia (PE) and preterm delivery (PTD) by analyzing extracellular vesicles (EVs) in maternal blood. EVs are small particles secreted by cells that carry proteins, RNA, and other biomolecules. They are known to play a key role in cell communication, including during pregnancy, especially between the placenta and maternal organs. The research focused on identifying specific surface markers found on EVs to help distinguish between pregnancies with complications and those without. The team developed a multiplex microarray slide embedded with antibodies targeting 17 different EV markers. They then tested maternal plasma samples using this platform without the need for purifying EVs, streamlining the diagnostic process.

CD24 is a mucin-like glycoprotein expressed on trophoblast cells and endothelial tissue of first and third trimester placentas. As an immune suppressor, CD24 may contribute to maternal immune tolerance to the growing fetus. CD24 is known to interact with the sialic acid-binding immunoglobulin-type lectins (Siglecs), specifically siglec-10. The aim of this study was to investigate the expression of both, CD24 and siglec-10 on placental tissue slides from acute covid patients, patients who survived a covid-19 infection and normal term controls. For the evaluation of CD24 & siglec-10 we used a total of 60 placentas, 10 acute covid-19 female, 10 acute covid-19 male, 10 post-covid-19 female, 10 post-covid-19 male, 10 female term controls and 10 male term controls. Immunohistochemical staining against CD24 and siglec-10 was performed and the expression of both markers was done with an immunoreactive score (IRS). Identity of CD24- or siglec-10 expressing cells was analyzed by double immune fluorescence analyses. The expression of CD24 is significantly downregulated on the extravillous trophoblast and on Hofbauer cells of female acute covid placentas. In the contrary, CD24 is significantly upregulated on male post-covid-19 Hofbauer cells. The CD24-ligand siglec-10 is significantly downregulated in post-covid-19 Hofbauer cells independently of fetal sex, whereas it shows significant higher expression in control female Hofbauer cells. CD24 and its ligand siglec-10 are differentially expressed in placentas of patients who survived a covid-19 infection. Surprisingly this effect is related to the fetal gender. Further investigation is necessary to analyze especially the imprinting effect of this infection.

At the beginning of the coronavirus disease 2019 (COVID-19) pandemic, uncertainties about the virus and its dangers during pregnancy caused great uncertainty and fear, especially among pregnant women. New data suggest an increased risk of obstetric complications, including maternal complications, preterm labor, intrauterine growth restriction, hypertensive disorders, stillbirths, gestational diabetes and risk, of neonatal developmental disorders. In addition, preeclampsia (PE)-like syndromes were also induced by severe COVID-19 infection. Therefore, the aim of this study was to investigate the expression of CD68 and CD163 and PD-L1 on placental tissues from acute covid patients, patients who survived a covid-19 infection and normal term controls that are known to be dysregulated in preeclampsia cases. We examined a total of 60 placentas from women that had given birth to female or male offspring in the University Hospital Augsburg. We investigated ten acute COVID-19 females, ten acute COVID-19 males, ten post-COVID-19 females, ten post-COVID-19 males, ten female term controls, and ten male term controls. Immunohistochemical staining against CD68, CD163, and PD-L1 was performed and the expression of the markers was evaluated with an immunoreactive score (percentage score). Identity of CD163- or PD-L1 expressing cells was analyzed by double immune fluorescence analyses. In opposite to PE, CD163 positive maternal macrophages are significantly upregulated in the decidua of male acute COVID-19 placentas. PD-L1 is significantly upregulated on male acute- and post-COVID-19 decidual immune cells and on male post-COVID-19 extravillous trophoblast cells. Surprisingly the observed effects are related to the fetal gender as they were not observed in female offsprings. Further investigation is necessary to analyze especially the imprinting effect of this infection.

INTRODUCTION:Placental-derived extracellular vesicles (EVs) are nano-organelles that facilitate intercellular communication between the feto-placental unit and the mother. We evaluated a novel Multiple Microarray analyzer for identifying surface markers on plasma EVs that predict preterm delivery and preeclampsia compared to term delivery controls. MATERIAL AND METHODS:In this prospective exploratory cohort study pregnant women between 24 and 40 gestational weeks with preterm delivery (n = 16), preeclampsia (n = 19), and matched term delivery controls (n = 15) were recruited from Bnai Zion Medical Center, Haifa, Israel. Plasma samples were tested using a multiple microarray analyzer. Glass slides with 17 antibodies against EV surface receptors - were incubated with raw plasma samples, detected by biotinylated secondary antibodies specific to EVs or placental EVs (PEVs), and labeled with cyanine 5-streptavidin. PBS and whole human IgG served as controls. The fluorescent signal ratio to negative controls was log 2 transformed and analyzed for sensitivity and specificity using the area under the receiver operating characteristics curves (AUROC). Best pair ratios of general EVs/PEVs were used for univariate analysis, and top pairs were combined for multivariate analysis. Results were validated by comparison with EVs purified using standard procedures. RESULTS:Heatmaps differentiated surface profiles of preeclampsia, preterm delivery, and term delivery receptors on total EVs and PEVs. Similar results were obtained with enriched EVs and EVs from raw plasma. Univariate analyses identified markers predicting preterm delivery and preeclampsia over term delivery controls with AUC >0.6 and sensitivity >50% at 80% specificity. Combining the best markers in a multivariate model, preeclampsia prediction over term delivery had an AUC of 0.89 (95% CI: 0.72-1.0) with 90% sensitivity and 90% specificity, marked by inflammation (TNF RII), relaxation (placenta protein 13 (PP13)), and immune-modulation (LFA1) receptors. Preterm delivery prediction over term delivery had an AUC of 0.97 (0.94-1.0), 84% sensitivity, and 90% specificity, marked by cell adhesion (ICAM), immune suppression, and general EV markers (CD81, CD82, and Alix). Preeclampsia prediction over preterm delivery had an AUC of 0.91 (0.79-0.99) with 80% sensitivity and 90% specificity with markers for complement activation (C1q) and autoimmunity markers. CONCLUSIONS:The new, robust EV Multi-Array analyzer and methodology offer a simple, fast diagnostic tool that reveals novel surface markers for major obstetric syndromes.

During pregnancy, an immune shield is created between the fetus and the mother to suppress fetal and placental rejection. CD24, a 30-70 kDa GPI- anchored and highly glycosylated cell surface protein, which can bind to P-selectin and Siglec-10 on haematopoitic cells. The CD24-Siglec-10 axix mediates immune tolerance, and, in association with signal transduction of DAMP, immunoregulation and anti-inflammatory effects. In solid tumours, the high expression of CD24 correlates with bad prognosis which can be explained by " Don't eat me" signals mediated by CD24-Siglec-10 axis [1]. First trimester CD24-Siglec-10 co-expression in close proximity to extravillous trophoblasts and endometrial glands, induces an immune suppression rendering the decidual invasion by the trophoblasts possible, as well as the blastocyst implantation in the uterine wall, thus avoiding maternal rejection of the pregnancy [2]. Previously, we explored the potential involvement of CD24 in immune protection. Initially, we have found that during the first trimester, CD24 is expressed in the maternal uterine glands, the villous and extravillous cytotrophoblasts, with a milder expression in stromal cells. While placental CD24 mRNA expression was increased from the first trimester to normal term controls by 2.27fold, a decreased expression was found in early preeclampsia cases as compared with unrelated preterm control and term deliveries. Lower labeling of the syncytiotrophoblasts was found in early preeclampsia compared to preterm and term control [3]. Altogether, a link between down regulation of CD24 and preeclampsia was implied. To gain more mechanistical insight into the CD24 link to preeclampsia, we studied its expression in cell lines trophoblast models reproducing some features of preeclampsia linked to over expression of the STOX-1 transcription factor, under its two major isoforms, STOX1A and STOX1B, that share the same gene target but have often opposite transcriptional effects [4]. Using qPCR, Western-Blot and flow cytometry, we studied CD24 in BeWo and JEG-3 cell lines stably overexpressing either of the two isoforms of the STOX1 transcription factor STOX 1A or STOX1B versus mock control CD24 gene expression was significantly down regulated by almost 90% in BeWo and ~70% in JEG-3 cells compared to the mock control by the expression or either STOX1A or STOX1B. Similar reduction level of total cellular CD24 protein was determined by Western Blotting in this in-vitro model. Flow cytometry analysis showed a somewhat lower level of reduction of cell surface expression of CD24, mainly in BeWo cells and less in JEG-3 cells. In all cases STOX1A was more potent in CD24 reduction than STOX1B isoform. In sum, using either actual preeclampsia placental samples or cell models mimicking some aspects of preeclampsia, an overall suppression of CD24 RNA and protein expression was demonstrated. Given the known link between CD24-Siglec-10 complex and immune suppression, we may hypothesize that CD24 reduction reflects a loss of immune tolerance in preeclampsia. Thus, attempts to elevate CD24 might be a novel innate immune checkpoint and a potential novel target for a possible immunotherapy of preeclampsia, a pathology where inflammation and cytokine play an important role, reminding the possible ways of alleviating the cytokine storm in COVID-19 infection.

Placental protein 13 (PP13) is a regulatory protein involved in remodeling the vascular system of the pregnancy and extending the immune tolerance of the mother to the growing fetus. PP13 is localized on the surface of the syncytiotrophoblast. An ex vivo placental model shows that the PP13 is released via placental-associated extracellular vesicles (PEVs) to the maternal uterine vein. This exploratory study aimed to determine PEV-associated PP13 in the maternal circulation as compared to the known soluble fraction since each has a specific communication pathway. Patients admitted to Bnai Zion Medical Center for delivery were recruited, and included 19 preeclampsia (PE) patients (7 preterm PE gestational age < 37 weeks' gestation), 16 preterm delivery (PTD, delivery at GA < 37 weeks' gestation), and 15 matched term delivery controls. Treatment by corticosteroids (Celestone), which is often given to patients with suspected preterm PE and PTD, was recorded. The PEV proteome was purified from the patients' plasma by size exclusion chromatography (SEC) to separate the soluble and PEV-associated PP13. The total level of PP13 (soluble and PEV-associated) was determined using mild detergent that depleted the PEV proteome. PP13 fractions were determined by ELISA with PP13 specific antibodies. ELISA with alkaline phosphatase (PLAP)- and cluster differentiation 63 (CD63)-specific antibodies served to verify the placental origin of the PEVs. SPSS was used for statistical analysis. The patients' medical, pregnancy, and delivery records in all groups were similar except, as expected, that a larger number of PE and PTD patients had smaller babies who were delivered earlier, and the PE patients had hypertension and proteinuria. The SEC analysis detected the presence of PP13 in the cargo of the PEVs and on their surface, in addition to the known soluble fraction. The median soluble PP13 was not significantly different across the PE, PTD, and term delivery control groups. However, after depleting the PEV of their proteome, the total PP13 (soluble and PEV-associated) was augmented in the cases of preterm PE, reaching 2153 pg/mL [IQR 1866-2838] but not in cases of PTD reaching 1576 pg/mL [1011-2014] or term delivery groups reaching 964 pg/mL [875-1636]), p < 0.01. On the surface of the circulating PEV from PTD patients, there was a decrease in PP13. Corticosteroid treatment was accompanied by a massive depletion of PP13 from the PEV, especially in preterm PE patients. This exploratory study is, thus, the first to determine PEV-associated PP13 in maternal circulation, providing a quantitative determination of the soluble and the PEV-associated fractions, and it shows that the latter is the larger. We found an increase in the amount of PP13 carried via the PEV-associated pathway in PE and PTD patients compared to term delivery cases, which was further augmented when the patients were treated with corticosteroids, especially in preterm PE. The signal conveyed by this novel communication pathway warrants further research to investigate these two differential pathways for the liberation of PP13.

Placental extracellular vesicles are delivered to maternal circulation acting as communication vehicles between the placenta and the maternal organs in various physiological and pathological conditions. We aim to determine the level of the immunoregulatory placental protein (PP13) in placental derived extracellular vesicles (pEV) in the maternal circulation in preeclampsia (PE) and preterm delivery (PTD) compared to term normal delivery. The cohort had 20 PE, 16 PTD and 14 matched term control cases. Extracellular vesicles were purified by size exclusion chromatography (SEC), characterized by CD63 and the placental origin of pEVs was verified by placental alkaline phosphatase. ELISA was used to quantify the level of soluble PP13, Its level in pEV and total PP13 determined after the pEV were depleted by plasma treatment by detergent. Using SEC, PP13 was determined on the PEV cargo in addition to its free level in the soluble plasma fraction. Total PP13 was elevated in the PE cases (P<0.01) compared to term normal deliveries, but not compared to PTD. On the isolated EVs, PP13 level was higher mainly in PE<37 weeks (P<0.01). Soluble form of PP13 was not different among the study and control group. Corticosteroid treatment was associated with increased release of PP13 to the plasma for both PE and PTD in either the total fraction or the EV aliquots This is the first study to determine PP13 as a part of the pEV proteome. Its level is increased in PE, which is further augmented among preterm PE treated with corticosteroids.

CD24 is a mucin-like immunosuppressing glycoprotein whose levels increase during pregnancy and decrease in the syncytio- and cytotrophoblasts in early and preterm preeclampsia. We used two modified cell lines that mimic in vitro features of preeclampsia to identify if this phenomenon could be reproduced. Our model was the immortalized placental-derived BeWo and JEG-3 cell lines that overexpress the STOX1 A/B transcription factor gene that was discovered in familial forms of preeclampsia. BeWo and JEG-3 cells stably transduced with the two major isoforms of STOX1-A/B or by an empty vector (control), were propagated, harvested, and analyzed. CD24 mRNA expression was determined by quantitative real-time polymerase nuclear chain reaction (qRT-PCR). CD24 protein levels were determined by Western blots. In STOX1-A/B overexpressing in BeWo cells, CD24 mRNA was downregulated by 91 and 85%, respectively, compared to the control, and by 30% and 74%, respectively in JEG-3 cells. A 67% and 82% decrease in CD24 protein level was determined by immunoblot in BeWo overexpressing STOX1-A/B, respectively, while the reduction in JEG-3 cells was between 47 and 62%. The immortalized BeWo and JEG-3 cell lines overexpressing STOX1-A/B had reduced CD24. Although both cell lines were affected, BeWo appears to be more susceptible to downregulation by STOX-1 than JEG-3, potentially because of their different cell origin and properties. These results strengthen the in vivo results of reduced CD24 levels found in early and preterm preeclampsia. Accordingly, it implies the importance of the reduced immune tolerance in preeclampsia, which was already demonstrated in vivo in the STOX1-A/B model of preeclampsia, and is now implied in the in vitro STOX-1 model, a subject that warrants further investigations.

KRAS mutations, which are the main cause of the pathogenesis of lethal pancreatic adenocarcinomas, impair the functioning of the GTPase subunit, thus rendering it constitutively active and signaling intracellular pathways that end with cell transformation. In the present study, the AsPC-1 cell line, which has a G12D-mutated KRAS gene sequence, was utilized as a cellular model to test peptide nucleic acid-based antisense technology. The use of peptide nucleic acids (PNAs) that are built to exhibit improved hybridization specificity and have an affinity for complementary RNA and DNA sequences, as well as a simple chemical structure and high biological stability that affords resistance to nucleases and proteases, enabled targeting of the KRAS-mutated gene to inhibit its expression at the translation level. Because PNA-based antisense molecules should be capable of binding to KRAS mRNA sequences, PNAs were utilized to target the mRNA of the mutated KRAS gene, a strategy that could lead to the development of a novel drug for pancreatic cancer. Moreover, it was demonstrated that introducing new PNA to cells inhibited the growth of cancer cells and induced apoptotic death and, notably, that it can inhibit G12D-mutated KRAS gene expression, as demonstrated by RT-PCR and western blotting. Altogether, these data strongly suggest that the use of PNA-based antisense agents is an attractive therapeutic approach to treating KRAS-driven cancers and may lead to the development of novel drugs that target the expression of other mutated genes.

Introduction: CD24 is a mucin-like glycoprotein expressed at the surface of hematopoietic and tumor cells and was recently shown to be expressed in the first trimester placenta. As it was postulated as an immune suppressor, CD24 may contribute to maternal immune tolerance to the growing fetus. Preeclampsia (PE), a major pregnancy complication, is linked to reduced immune tolerance. Here, we explored the expression of CD24 in PE placenta in preterm and term cases. Methods: Placentas were derived from first and early second trimester social terminations (N = 43), and third trimester normal term delivery (N = 67), preterm PE (N = 18), and preterm delivery (PTD) (N = 6). CD24 expression was determined by quantitative polymerase chain reaction (qPCR) and Western blotting. A smaller cohort included 3–5 subjects each of term and early PE, and term and preterm delivery controls analyzed by immunohistochemistry. Results: A higher expression (2.27-fold) of CD24 mRNA was determined in the normal term delivery compared to first and early second trimester cases. The mRNA of preterm PE cases was only higher by 1.31-fold compared to first and early second trimester, while in the age-matched PTD group had a fold increase of 5.72, four times higher compared to preterm PE. The delta cycle threshold (ΔCt) of CD24 mRNA expression in the preterm PE group was inversely correlated with gestational age (r = 0.737) and fetal size (r = 0.623), while correlation of any other group with these parameters was negligible. Western blot analysis revealed that the presence of CD24 protein in placental lysate of preterm PE was significantly reduced compared to term delivery controls (p = 0.026). In immunohistochemistry, there was a reduction of CD24 staining in villous trophoblast in preterm PE cases compared to gestational age-matched PTD cases (p = 0.042). Staining of PE cases at term was approximately twice higher compared to preterm PE cases (p = 0.025) but not different from normal term delivery controls. Conclusion: While higher CD24 mRNA expression levels were determined for normal term delivery compared to earlier pregnancy stages, this expression level was found to be lower in preterm PE cases, and could be said to be linked to reduced immune tolerance in preeclampsia.

CD24 is a highly glycosylated protein with a small protein core that is linked to the plasma membrane via a glycosyl-phosphatidylinositol anchor. CD24 is primarily expressed by immune cells but is often overexpressed in human tumors. In cancer, CD24 is a regulator of cell migration, invasion and proliferation. Its expression is associated with poor prognosis and it is used as cancer stemness marker. Recently, CD24 on tumor cells was identified as a phagocytic inhibitor ("do not eat me" signal) having a suppressive role in tumor immunity via binding to Siglec-10 on macrophages. This finding is reminiscent of the demonstration that soluble CD24-Fc can dampen the immune system in autoimmune disease. In the present review, we summarize recent progress on the role of the CD24-Siglec-10 binding axis at the interface between tumor cells and the immune system, and the role of CD24 genetic polymorphisms in cancer. We describe the specific function of cytoplasmic CD24 and discuss the presence of CD24 on tumor-released extracellular vesicles. Finally, we evaluate the potential of CD24-based immunotherapy.

INTRODUCTION:In a recent study of 10,011 pregnant women, 95% of miscarriages occurred before routine ultrasound scan at 11-14 weeks. Our study aimed to identify early first trimester parameters which may predict miscarriage before 10 weeks of gestation for in vitro fertilization (IVF) pregnancies. METHODS:A cohort of 115 healthy IVF patients with a singleton viable embryo in early first trimester were studied in a tertiary university-affiliated medical center (April 2017-June 2018). Calculations included gestational age (GA); ultrasound evaluation of crown-rump length (CRL), mean gestational sac diameter (GSD) and volume (GSV), mean yolk sac diameter (YSD) and volume (YSV); fetal heart rate (FHR), mean uterine arteries pulsatility index (UtA-PI); and maternal blood placental protein 13 (PP13) levels. Patients were divided into three groups by GA; and early miscarriage versus ongoing pregnancy after GA 10 weeks. RESULTS:Early fetal loss occurred in 14.8% of patients; miscarriage group had higher discrepancy between calculated and measured GA (P < 0.001), lower GSD and GSV (P = 0.005 and P = 0.02, respectively), significantly different YSD and YSV, and lower GSD/YSD and GSV/YSV ratios (P = 0.001 and P = 0.003, respectively). UtA-PI/CRL ratio was higher in patients with miscarriage at GA 46-48 days and GA >48 days (P = 0.034 and P = 0.026, respectively). PP13/CRL ratio was higher in patients with miscarriage at GA >48 days (P = 0.041). DISCUSSION:In IVF pregnancies with live embryo at first ultrasound scan, high UtA-PI/CRL and maternal blood PP13/CRL ratios may indicate impaired placentation preceded early pregnancy loss. A larger cohort is needed to further verify these predictions.