The Role of Extracellular Vesicles (EVs) in Chronic Graft vs. Host Disease, and the Potential Function of Placental Cell-Derived EVs as a Therapeutic Tool.

Chronic graft-versus-host disease (cGVHD) presents with dermal inflammation and fibrosis. We investigated the characteristics of extracellular vesicles (EVs) obtained from cGVHD patients, and their potential effects on human dermal fibroblast (NHDF) cells. The anti-inflammatory and anti-fibrotic effects of placental EVs were also explored given their known anti-inflammatory properties. Fourteen cGVHD patients' EVs contained higher levels of fibrosis-related proteins, TGFß and α-smooth muscle actin (αSMA), compared to EVs from thirteen healthy subjects. The exposure of NHDF cells to the patients' EVs increased the NHDF cells' TGFß and αSMA expressions. Placental EVs derived from placental-expanded cells (PLX) (Pluri Inc.) and human villous trophoblast (HVT) cells expressing the mesenchymal markers CD29, CD73, and CD105, penetrated into both the epidermal keratinocytes (HACATs) and NHDF cells. Stimulation of the HACAT cells with cytokine TNFα/INFγ (0.01-0.1 ng/µL) reduced cell proliferation, while the addition of placental EVs attenuated this effect, increasing and normalizing cell proliferation. The treatment of NHDF cells with a combination of TGFß and placental HVT EVs reduced the stimulatory effects of TGFß on αSMA production by over 40% (p = 0.0286). In summary, EVs from patients with cGVHD can serve as a biomarker for the cGVHD state. Placental EVs may be used to regulate dermal inflammation and fibrosis, warranting further investigation of their therapeutic potential.

Extracellular Vesicles of COVID-19 Patients Reflect Inflammation, Thrombogenicity, and Disease Severity.

Severe COVID-19 infections present with cytokine storms, hypercoagulation, and acute respiratory distress syndrome, with extracellular vesicles (EVs) being involved in coagulation and inflammation. This study aimed to determine whether coagulation profiles and EVs reflect COVID-19 disease severity. Thirty-six patients with symptomatic COVID-19 infection with mild/moderate/severe disease (12 in each group) were analyzed. Sixteen healthy individuals served as controls. Coagulation profiles and EV characteristics were tested by nanoparticle tracking analysis (NTA), flow cytometry, and Western blot. While coagulation factors VII, V, VIII, and vWF were comparable, significant differences were found in patients' D-Dimer/fibrinogen/free protein S levels compared to controls. Severe patients' EVs displayed higher percentages of small EVs (<150 nm) with increased expression of exosome marker CD63. Severe patients' EVs displayed high levels of platelet markers (CD41) and coagulation factors (tissue factor activity, endothelial protein C receptor). EVs of patients with moderate/severe disease expressed significantly higher levels of immune cell markers (CD4/CD8/CD14) and contained higher levels of IL-6. We demonstrated that EVs, but not the coagulation profile, may serve as biomarkers for COVID-19 severity. EVs demonstrated elevated levels of immune- and vascular-related markers in patients with moderate/severe disease, and may play a role in disease pathogenesis.

Time of Birth and the Risk of Adverse Maternal and Neonatal Outcomes-A Retrospective Cohort Study.

Objectives: To determine whether in a labor floor housed continuously by senior physicians the risk of adverse maternal and neonatal outcome is affected by time of delivery. Methods: This retrospective cohort study, conducted at a tertiary medical center, assessed singleton term deliveries from 1 January 2011 to 30 January 2020. Participants were categorized based on delivery timing, correlating with nursing shifts, to evaluate perinatal outcomes. The primary endpoint included adverse maternal outcomes such as emergency Cesarean section, anal sphincter injuries, blood product transfusions, and postpartum surgeries (laparotomy/laparoscopy). Secondary outcomes focused on neonatal health indicators, including low Apgar scores, ICU admissions, respiratory issues, extended hospital stays, and neurological complications. Results: 87,863 deliveries were available for analysis with equal distribution during the day. The risk of adverse composite maternal outcome was highest during the evening (aOR 1.25, 95% CI 1.18-1.32) and lowest during the night (aOR 0.94, 95% CI 0.88-0.99) compared to daytime deliveries. This difference was primarily driven by the highest rate of emergency CD in the evening. Neonatal outcomes were comparable, except for length of stay > 5 days, which was more frequent among newborns delivered during the evening and night shifts compared to the morning shift (aOR 1.19, 95% CI 1.07-1.33 and aOR 1.17, 95% CI 1.05-1.31, respectively). Conclusions: In term pregnancies, the evening shift is associated with the highest risk of adverse maternal and neonatal outcomes despite physician seniority.

Characterization of extracellular vesicles in COVID-19 infection during pregnancy.

Introduction: SARS-CoV-2 infection may cause a severe inflammatory response, inflicting severe morbidity and mortality. This risk is modestly increased in pregnant patients. Despite the hypercoagulability and immunosuppression associated with pregnancy, most pregnant women experience a mild COVID-19 infection. Maternal extracellular vesicles (EVs) may interact with endothelial and immune components to facilitate a favorable disease course. This pilot study aimed to explore the characteristics of EVs released during COVID-19 infection occurring during the third trimester of pregnancy. Methods: In this prospective study, blood samples were obtained from 16 healthy non-pregnant (NP), 18 healthy-pregnant (HP), and 22 COVID-19 positive pregnant subjects (CoV-P). Disease course and pregnancy outcomes were assessed and EVs were characterized. Of note, limited volumes of sample acquired from the subjects made it necessary to use smaller and different subsets of samples for each analysis. Results: The majority (91%) of the COVID-19-pregnant subjects (18 mild and 2 moderate disease) experienced good pregnancy-related outcomes. EV concentrations were higher in healthy-pregnant subjects compared to non-pregnant subjects (p = 0.0041) and lower in COVID-19-pregnant subjects compared to healthy-pregnant subjects (p = 0.0150). CD63 exosome marker expression was higher in EVs of healthy-pregnant subjects and COVID-19-pregnant subjects compared to EVs of non-pregnant subjects (p = 0.0149, p = 0.0028, respectively). Similar levels of SARS-CoV-2 entry proteins (ACE-2 and TMPRSS2) were found in all three groups. Cytokine content increased in healthy-pregnant subject-EVs compared to non-pregnant EVs, while IL-2 and IL-6 levels were decreased in COVID-19-pregnant subject-EVs compared to healthy-pregnant subject-EVs (p = 0.043, p = 0.0390, respectively). CD8+, cytotoxic T-cell marker, was lower in non-pregnant EVs compared to healthy-pregnant subject-EVs and to COVID-19-pregnant subjects (p = 0.0108, p < 0.0001, respectively). COVID-19- pregnant subject-EVs demonstrated higher levels of platelet activation marker (CD62P) than non-pregnant (p = 0.0327) and healthy-pregnant subjects (p = 0.0365). Endothelial marker EV-CD144+ was lower in healthy-pregnant subjects versus non-pregnant subjects (p = 0.0093), but similar in COVID-19-pregnant and non-pregnant subjects. Other EVs' coagulation markers/activity, D-Dimer and fibrinogen levels were similar in healthy-pregnant subjects and COVID-19 positive pregnant subjects. Conclusion: COVID-19 positive pregnant subjects' EVs demonstrated an attenuated inflammatory response, with no additional activation of the coagulation system.

Associations of maternal and placental extracellular vesicle miRNA with preeclampsia.

Introduction: Gestational vascular complications (GVCs), including gestational hypertension and preeclampsia, are leading causes of maternal morbidity and mortality. Elevated levels of extracellular vesicles (EVs), in GVC have been linked to vascular injury. This study aims to characterize placental and circulating EV miRNA in GVCs, and explores the involvement of EV-miRNA in GVC, and whether they may be used to distinguish between placental and maternal pathologies. Methods: Blood samples were obtained from 15 non-pregnant (NP), 18 healthy-pregnant (HP), and 23 women with GVC during the third trimester. Placental sections were obtained after caesarian section. Platelet-poor-plasma (PPP) and EV pellets were characterized: EV size/concentration, protein content and miRNA expression were measured by nanoparticle tracking analysis, western blot, nano-string technology and RT-PCR. The effects of EVs on trophoblasts and EC miRNA expression were evaluated. Results: Higher EVs concentrations were observed in HP-PPP and GVC-PPP (p < 0.0001) compared to the NP-PPP. The concentration of large EVs (>100 nm) was higher in PPP and EV pellets of HP and GVC compared to the NP group. EV pellets of pregnant women demonstrated lower expression of exosomal markers CD63/CD81 compared to NP-EVs. GVC-EVs expressed more human placental lactogen (hPL) hormone than HP-EVs, reflecting their placental origin. Screening of miRNAs in EV pellets and in PPP identified certain miRNAs that were highly expressed only in EVs pellets of the HP (13%) and GVC groups (15%), but not in the NP group. Differences were detected in the expression of hsa-miR-16-5p, hsa-miR-210, and hsa-miR-29b-3p. The expression of hsa-miR-16-5p and hsa-miR-210 was low in EV pellets obtained from NP, higher in HP-EVs, and significantly lower in GVC-EVs. Except for hsa-miR-29b-3p, which was upregulated in GVC, no significant differences were found in the levels of other miRNAs in placental sections. Exposure to GVC-EVs resulted in higher expression of hsa-miR-29b-3p compared to cells exposed to HP-EVs in villous trophoblasts, but not in EC. Conclusion: Expression of hsa-miR-16-5p and hsa-miR-210 reflects maternal pathophysiological status, while hsa-miR-29b-3p reflects placental status. These findings suggest that EV-miRNA are involved in GVC, and that they may be used to distinguish between pathologies of placental and maternal origins in preeclampsia.

The effects of time and temperature on umbilical cord gas analysis.

OBJECTIVE: Our objective was to evaluate the effects of time and temperature on umbilical-cord blood analysis. METHODS: This prospective study included the term spontaneous vaginal deliveries. One venous and seven arterial samples were drawn from each umbilical cord within 5 min from delivery. Three samples were immediately refrigerated (3 °C), while all other samples were stored at room temperature (23-26 °C). Samples were analyzed in pairs (refrigerated and room-temperature samples) at 0, 20, 40, and 60 min after delivery for pH and lactate levels. Repeated-measures analysis using a generalized linear model was used to compare the change in pH and lactate values over time. RESULTS: 518 samples from 74 women were analyzed. The mean gestational age was 39.1 ± 1.1 weeks. All neonates had an Apgar score of ≥9 in the 1st and 5th minutes. Mean arterial pH and lactate levels at delivery (time 0) were 7.32 ± 0.07 and 4.00 ± 1.36 mmol/L, respectively. Over time, a statistically significant decrease in pH and a reciprocal increase in lactate levels were observed. The mean change in arterial pH following 60 min was 0.021 ± 0.028 (room-temperature) and 0.016 ± 0.023 (refrigerated); p < 0.001. Compared to pH, a greater change was demonstrated in lactate levels over time; the mean change in lactate following 60 min was -0.896 ± 0.535 (room temperature) and -0.512 ± 0.450 mmol/L (refrigerated). Temperature significantly altered both pH and lactate levels, but lactate levels were altered at earlier time points. CONCLUSION: Both time and temperature have significant effects on cord blood analysis. Yet, these changes are minor and may not have any clinical significance unless in extreme cases in which medicolegal aspects emerge.

Abdominal computed tomography (CT) scan in the evaluation of refractory puerperal fever: impact on management.

Introduction: Computed tomography (CT) imaging should be employed judiciously, given its cost, use of intravenous contrast, and ionizing radiation. The aim of this study was to determine the clinical benefit of a CT scan in the evaluation of refractory puerperal fever and to identify the appropriate candidates for its use.Methods: This was a retrospective cohort study conducted in a single tertiary care center between January 2007 to April 2017. Indications for CT scan were refractory postpartum fever of ≥3 days and/or ultrasound findings suggesting complex abdominal fluid collection. Primary outcome was defined as a change in the mode of treatment due to the CT findings. In addition, a multivariate analysis of risk factors for puerperal fever was performed to identify patients who would benefit from the CT scan evaluation.Results: There were 520 women that underwent an abdominal and pelvic CT scan during the study period, 238 (45.7%) met inclusion criteria, 94 (39.5%) had a normal CT scan, and 144 (60.5%) had abnormal findings including 32 (13.4%) cases with pelvic thrombophlebitis and 112 (47%) cases with pelvic fluid collections. Results of the CT changed clinical management in 93 (39.0%) patients, including: switching antibiotics in 24 (10%) patients, adding low molecular weight heparin for 28 (11.8%) patients, and surgical intervention (laparotomy or drainage insertion) in 41 (17.2%) patients. In the regression model, we didn't find any significant risk factors associated with treatment change following the CT scan.Conclusions: Abdominal and pelvic CT scan in women with refractory puerperal fever has a high clinical yield and lead to a change in management in a substantial number of patients.