Exosomes/EVs: IMMUNOMODULATORY EFFECTS OF AMNITOIC FLUID EXTRACELLULAR VESICLES IN AN IN-VITRO MODEL OF IMMUNE ACTIVATION

Background & Aim: Amniotic fluid (AF)-derived EVs are currently under investigation for use as anti-inflammatory therapeutics in COVID-19 and COVID-19 long haulers. The dysregulation of the immune response induced by SARS-COV-2 is a key driver of both acute COVID-19 induced lung injury and long term COVID-19 sequela. There is a clear need to identify therapeutics that suppress excessive inflammation and reduce immune cell exhaustion to improve patient short term and long-term outcomes. Amniotic fluid (AF)- derived extracellular vesicles (EVs) have previously been shown to deliver anti-inflammatory and immune-modulatory signals to diverse cellular targets. We aimed to test if AF-EVs carry immune-suppressive molecules and can suppress T-cell immune activation and exhaustion in vitro. Methods, Results & Conclusion(s): The AF-EV biologic tested was derived from AF collected from consenting donors during planned, fullterm cesarean sections. AF was centrifuged and filtered to remove cellular debris and create a product containing AF-EVs and soluble extracellular components. Fluorescent EXODOT analysis was performed to demonstrate the presence of EV markers CD9, CD81, ALIX, and immune suppressive molecule PD-L1. T-cell activation/exhaustion was induced in vitro by treating human peripheral blood mononuclear cells with activation agent PHA for 3 days with the addition of AF-EVs or saline control. Immune activation/exhaustion was measured by flow cytometry to determine the expression of PD-1 on CD3+ T-cells. The AF-EV biologic was characterized to contain EVs with positive expression of CD9, CD81, ALIX, and PL-L1. T-cell activation/exhaustion was upregulated in response to PHA and was significantly reduced by 8% in AF-EV treated T-cells compared to saline control (77.7% vs 85.7%, respectively P<0.05). These findings demonstrate that AF-EVs do express PD-L1, a surface marker that has previously been demonstrated to contribute to exosome-mediated immunosuppression. Furthermore, we confirmed in vitro that AF-EVs suppress T-cell activation/ exhaustion in the presence of a T-cell activation agent. COVID-19 long haulers have been described to have upregulated and pro-longed immune activation and T-cell exhaustion, marked by an increase in PD1+ T-cells. Therefore, this finding serves as a starting point for the development of a potential mechanism of action that may describe AF-EV's therapeutic effect in COVID-19 long hauler patients.Copyright © 2023 International Society for Cell & Gene Therapy

Obstetric Disorders and Critical Illness.

In this article, we discuss some of the more common obstetric-related conditions that can lead to critical illness and require management in an ICU. These include the hypertensive disorders of pregnancy, postpartum hemorrhage, hemolysis, elevated liver enzymes, and low platelet syndrome, acute fatty liver of pregnancy, amniotic fluid embolism, and peripartum cardiomyopathy. We also discuss pulmonary embolism and Covid-19. Despite not being specific to obstetric patients, pulmonary embolism is a common, life-threatening diagnosis in pregnancy with particular risks and management aspects. Covid-19 does not seem to occur with higher frequency in pregnant women, but it leads to higher rates of ICU admissions and mechanical ventilation in pregnant women than in their nonpregnant peers. Its prevalence during our current global pandemic makes it important to discuss in this article. We provide a basis for critical care physicians to be engaged in informed conversations and management in a multidisciplinary manner with other relevant providers in the care of critically ill pregnant and postpartum women.

The incidence and risk factors of meconium amniotic fluid in singleton pregnancies: an experience of a tertiary hospital in Iran.

BACKGROUND: Several common maternal or neonatal risk factors have been linked to meconium amniotic fluid (MAF) development; however, the results are contradictory, depending on the study. This study aimed to assess the prevalence and risk factors of MAF in singleton pregnancies. METHODS: This study is a retrospective cohort that assessed singleton pregnant mothers who gave birth at a tertiary hospital in Bandar Abbas, Iran, between January 1st, 2020, and January 1st, 2022. Mothers were divided into two groups: 1) those diagnosed with meconium amniotic fluid (MAF) and 2) those diagnosed with clear amniotic fluid. Mothers with bloody amniotic fluid were excluded. Demographic factors, obstetrical factors, and maternal comorbidities were extracted from the electronic data of each mother. The Chi-square test was used to compare differences between the groups for categorical variables. Logistic regression models were used to assess meconium amniotic fluid risk factors. RESULTS: Of 8888 singleton deliveries during the study period, 1085 (12.2%) were MAF. MAF was more common in adolescents, mothers with postterm pregnancy, and primiparous mothers, and it was less common in mothers with GDM and overt diabetes. The odds of having MAF in adolescents were three times higher than those in mothers 20-34 years old (aOR: 3.07, 95% CI: 1.87-4.98). Likewise, there were significantly increased odds of MAF in mothers with late-term pregnancy (aOR: 5.12, 95% CI: 2.76-8.94), and mothers with post-term pregnancy (aOR: 7.09, 95% CI: 3.92-9.80). Primiparous women were also more likely than multiparous mothers to have MAF (aOR: 3.41, 95% CI: 2.11-4.99). CONCLUSIONS: Adolescents, primiparous mothers, and mothers with post-term pregnancies had a higher risk of MAF. Maternal comorbidities resulting in early termination of pregnancy can reduce the incidence of MAF.

Outcome of Newborns with Confirmed or Possible SARS-CoV-2 Vertical Infection-A Scoping Review.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2), the virus that causes 2019 coronavirus disease (COVID-19), has been isolated from various tissues and body fluids, including the placenta, amniotic fluid, and umbilical cord of newborns. In the last few years, much scientific effort has been directed toward studying SARS-CoV-2, focusing on the different features of the virus, such as its structure and mechanisms of action. Moreover, much focus has been on developing accurate diagnostic tools and various drugs or vaccines to treat COVID-19. However, the available evidence is still scarce and consistent criteria should be used for diagnosing vertical transmission. Applying the PRISMA ScR guidelines, we conducted a scoping review with the primary objective of identifying the types, and examining the range, of available evidence of vertical transmission of SARS-CoV-2 from mother to newborn. We also aimed to clarify the key concepts and criteria for diagnosis of SARS-CoV-2 vertical infection in neonates and summarize the existing evidence and advance the awareness of SARS-CoV-2 vertical infection in pregnancy. Most studies we identified were case reports or case series (about 30% of poor quality and inconsistent reporting of the findings). Summarizing the existing classification criteria, we propose an algorithm for consistent diagnosis. Registration: INPLASY2022120093.

ALTERED AMINO ACID PROFILES OF THE "MOTHER-FETUS" SYSTEM IN COVID-19

Systemic nature of the human body response to SARS-CoV-2 requires dedicated analysis at the molecular level. COVID-19 during pregnancy affects maternal health and may entail complications in the early neonatal period and possibly long-term consequences for the offspring. The aim of the study was to assess the impact of COVID-19 on amino acid profiles in maternal venous blood, amniotic fluid and umbilical cord blood in order to develop a diagnostic panel accounting for possible consequences. The main group included 29 pregnant patients with a confirmed diagnosis of COVID-19 and the control group included 17 somatically healthy pregnant women. Amino acid profiles of the biological fluids were measured by high-performance liquid chromatography combined to mass spectrometry (HPLC-MS) and assessed in logistic regression models. The analysis revealed altered content of certain amino acids, their biosynthetic precursors and metabolites in the biological fluids collected from patients with COVID-19 possibly reflecting the development of systemic inflammatory reaction and associated changes in gene expression profiles. These findings may guide further research into health outcomes for neonates born from mothers infected with SARS-CoV-2 during pregnancy. The study may help to develop advanced recommendations and differential care protocols for pregnant women and newborns diagnosed with COVID-19.

Evidence of Maternal Antibodies Elicited by COVID-19 Vaccination in Amniotic Fluid: Report of Two Cases in Italy.

With SARS-CoV-2 infection, pregnant women may be at a high risk of severe disease and adverse perinatal outcomes. A COVID-19 vaccination campaign represents the key strategy to combat the pandemic; however, public acceptance of maternal immunization has to be improved, which may be achieved by highlighting the promising mechanism of passive immunity as a strategy for protecting newborns against SARS-CoV-2 infection. We tested the anti-SARS-CoV-2 antibody response following COVID-19 full-dose vaccination in the serum and amniotic fluid of two pregnant women who presented between April and June 2021, at the Center for the Treatment and Prevention of Infections in Pregnancy of the National Institute for Infectious Diseases "L. Spallanzani", for antenatal consultancy. Anti-SARS-CoV-2 IgG was found in residual samples of amniotic fluid collected from both women at the 18th week of gestation (63 and 131 days after the second dose's administration). Titers in amniotic fluid mirrored the levels detected in serum and were inversely linked to the time from vaccination. Our results suggest that antibodies elicited by COVID-19 vaccination can cross the placenta and reach the fetus; therefore, they may offer passive immunity at birth. It is critical to fully understand the kinetics of the maternal response to vaccination, the efficiency of IgG transfer, and the persistence of antibodies in infants to optimize maternal immunization regimens.

Probable congenital SARS-CoV-2 infection in a neonate born to a woman with active SARS-CoV-2 infection: CMAJ

Kirtsman et al discuss the probable congenital SARS-CoV-2 infection in a neonate born to a woman with active SARS-CoV-2 infection. They present a case study of a 40-year-old woman who was admitted to a tertiary hospital in Toronto, Ontario. She had familial neutropenia, gestational diabetes and a history of frequent bacterial infections during pregnancy, which resolved with antibiotic treatment. Details of the maternal course and outcome have been published separately because of her hematologic condition. A nasopharyngeal swab was positive for suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene targets via reverse transcription polymerase chain reaction (RT-PCR) testing. There were no fetal concerns during the pregnancy or following admission. A semiurgent cesarean delivery was done under regional anesthesia, with airborne, droplet and contact precautions, owing to worsening coagulopathy and reducing platelet count at 35 weeks. Artificial rupture of membranes was performed at operation. The male neonate was vigorous and did not require resuscitation. His Apgar scores were 9 at 1 minute and 9 at 5 minutes, but all 3 of the neonate's nasopharyngeal swabs were positive for SARS-CoV-2 gene targets via RT-PCR testing.

Potential effect of amniotic fluid-derived stem cells on hyperoxia-induced pulmonary alveolar injury.

BACKGROUND: With the widespread of Coronavirus Disease 2019 pandemic, in spite of the newly emerging vaccines, mutated strains remain a great obstacle to supportive and preventive measures. Coronavirus 19 survivors continue to face great danger of contacting the disease again. As long as no specific treatment has yet to be approved, a great percentage of patients experience real complications, including among others, lung fibrosis. High oxygen inhalation especially for prolonged periods is per se destructive to the lungs. Nevertheless, oxygen remains the first line support for such patients. In the present study we aimed at investigating the role of amniotic fluid-mesenchymal stem cells in preventing versus treating the hyperoxia-induced lung fibrosis in rats. METHODS: The study was conducted on adult albino rats; 5 pregnant female rats were used as amniotic fluid donors, and 64 male rats were randomly divided into two groups: Control group; where 10 rats were kept in normal atmospheric air then sacrificed after 2 months, and hyperoxia-induced lung fibrosis group, where 54 rats were exposed to hyperoxia (100% oxygen for 6 h/day) in air-tight glass chambers for 1 month, then randomly divided into the following 5 subgroups: Hyperoxia group, cell-free media-treated group, stem cells-prophylactic group, stem cells-treated group and untreated group. Isolation, culture and proliferation of stem cells were done till passage 3. Pulmonary function tests, histological examination of lung tissue under light and electron microscopes, biochemical assessment of oxidative stress, IL-6 and Rho-A levels, and statistical analysis of data were performed. F-test (ANOVA) was used for normally distributed quantitative variables, to compare between more than two groups, and Post Hoc test (Tukey) for pairwise comparisons. RESULTS: Labelled amniotic fluid-mesenchymal stem cells homed to lung tissue. Stem cells administration in the stem cells-prophylactic group succeeded to maintain pulmonary functions near the normal values with no significant difference between their values and those of the control group. Moreover, histological examination of lung tissues showed that stem cells-prophylactic group were completely protected while stem cells-treated group still showed various degrees of tissue injury, namely; thickened interalveolar septa, atelectasis and interstitial pneumonia. Biochemical studies after stem cells injection also showed decreased levels of RhoA and IL-6 in the prophylactic group and to a lesser extent in the treated group, in addition to increased total antioxidant capacity and decreased malondialdehyde in the stem cells-injected groups. CONCLUSIONS: Amniotic fluid-mesenchymal stem cells showed promising protective and therapeutic results against hyperoxia-induced lung fibrosis as evaluated physiologically, histologically and biochemically.

Small Extracellular Vesicles from Human Amniotic Fluid Samples as Promising Theranostics.

Since the first evidence that stem cells can provide pro-resolving effects via paracrine secretion of soluble factors, growing interest has been addressed to define the most ideal cell source for clinical translation. Leftover or clinical waste samples of human amniotic fluid obtained following prenatal screening, clinical intervention, or during scheduled caesarean section (C-section) delivery at term have been recently considered an appealing source of mesenchymal progenitors with peculiar regenerative capacity. Human amniotic fluid stem cells (hAFSC) have been demonstrated to support tissue recovery in several preclinical models of disease by exerting paracrine proliferative, anti-inflammatory and regenerative influence. Small extracellular vesicles (EVs) concentrated from the hAFSC secretome (the total soluble trophic factors secreted in the cell-conditioned medium, hAFSC-CM) recapitulate most of the beneficial cell effects. Independent studies in preclinical models of either adult disorders or severe diseases in newborns have suggested a regenerative role of hAFSC-EVs. EVs can be eventually concentrated from amniotic fluid (hAF) to offer useful prenatal information, as recently suggested. In this review, we focus on the most significant aspects of EVs obtained from either hAFSC and hAF and consider the current challenges for their clinical translation, including isolation, characterization and quantification methods.

Assessment of materno-foetal transmission of SARS-CoV-2: A prospective pilot study.

BACKGROUND: The issue of vertical transmission of SARS-CoV-2 infection to the foetus has not yet been resolved. Its main reason is lack of a bigger study to analyse this question. The evidence of the affection of the foetus during antenatal or intrapartum period is limited to some anecdotal reports. To look for the possibility of vertical transmission of Severe Acute Respiratory Syndrome - Corona Virus-2 (SARS-CoV-2) infection to the foetus, this prospective pilot study was conducted at a tertiary health care COVID-19 designated centre of Armed Forces. METHODS: This study was conducted during 01 June 2020 and 15 October 2020 and included 54 covid-positive pregnant mothers. During delivery, amniotic fluid and cord blood samples were collected in a sterile manner. Amniotic fluid samples were not collected during vaginal deliveries as chances of contamination was very high. These samples were tested for the presence of SARS-CoV-2 gene by Reverse Transcriptasee Polymerase Chain Reaction (RT-PCR) test, and the results were analysed. Newborns were allowed to room in with mother, and they underwent throat and nasal swab RT-PCR testing of covid within 24-48 h of delivery. RESULTS: A total of 1520 pregnant mothers underwent RT-PCR test during the study period. Total positivity rate among our pregnant women was 2.8%. Out of 54 covid-positive women during the study period, amniotic fluid RT-PCR tests were carried out for 43 women, and cord blood was tested for 45 women. CONCLUSION: RT-PCR test of amniotic fluid, cord blood and nasal and throat swab of all newborns delivered by SARS-CoV-2-positive pregnant women were negative. Based on our study, the possibility of intrauterine vertical transmission of SARS-CoV-2 infection appears to be unlikely.

Case Report: Administration of Amniotic Fluid-Derived Nanoparticles in Three Severely Ill COVID-19 Patients.

Rationale/Objectives: A human coronavirus (HCoV-19) has caused the novel coronavirus disease (COVID-19) outbreak worldwide. There is an urgent need to develop new interventions to suppress the excessive immune response, protect alveolar function, and repair lung and systemic organ damage. Zofin (previously known as Organicell Flow) is a novel therapeutic that is derived from the soluble and nanoparticle fraction (extracellular vesicles and exosomes) of human amniotic fluid. Here within, we present the clinical outcomes after Zofin treatment in three critically ill patients suffering from severe, multi-organ complications induced by COVID-19 infection. All patients were diagnosed with COVID-19, developed respiratory failure, and were hospitalized for more than 40 days. Methods: Zofin was administered to patients concurrently with ongoing medical care who were monitored for 28-days post-therapy. SOFA score assessment, chest X-rays, and inflammatory biomarker testing was performed. Main Results: There were no adverse events associated with the therapy. The patients showed improvements in ICU clinical status and experienced respiratory improvements. Acute delirium experienced by patients completely resolved and inflammatory biomarkers improved. Conclusions: Primary outcomes demonstrate the therapy was safe, accessible, and feasible. This is the first demonstration of human amniotic fluid-derived nanoparticles as a safe and potentially efficacious therapeutic treatment for respiratory failure induced by COVID-19 infection.

Does the maternal-fetal transmission of SARS-CoV-2 occur during pregnancy? / ¿Existe transmisión materno-fetal del SARS-CoV-2 durante la gestación?

BACKGROUND AND OBJECTIVE: On January 7th, 2020, a new coronavirus, SARS-CoV-2, was identified, as responsible for a new human disease: COVID-19. Given its recent appearance, our current knowledge about the possible influence that this disease can exert on pregnancy is very limited. One of the unknowns to be solved is whether there is a vertical transmission of the infection during pregnancy. PATIENTS AND METHODS: Using the Real-time Polymerase Chain Reaction techniques for SARS-CoV-2 nucleic acids, the possible presence of this germ in vaginal discharge and amniotic fluid was investigated in four pregnant Caucasian patients affected by mild acute symptoms of COVID-19 during the second trimester of pregnancy. RESULTS: There is no laboratory evidence to suggest a possible passage of SARS-CoV-2 from the infected mother to the amniotic fluid. CONCLUSIONS: It is necessary to expand the investigation of COVID-19 cases diagnosed during pregnancy to clarify the real influence that SARS-CoV-2 has on pregnant women and their offspring, as well as those factors that modulate the disease.

Gestation and COVID-19: clinical and microbiological observational study (Gesta-COVID19).

BACKGROUND: The Coronavirus Disease 2019 (COVID-19) is a novel disease which has been having a worldwide affect since December 2019. Evidence regarding the effects of SARS-CoV-2 during pregnancy is conflicting. The presence of SARS-CoV-2 has been demonstrated in biological samples during pregnancy (placenta, umbilical cord or amniotic fluid); however, maternal and fetal effects of the virus are not well known. METHODS: Descriptive, multicentre, longitudinal, observational study in eight tertiary care hospitals throughout Spain, that are referral centres for pregnant women with COVID-19. All pregnant women with positive SARS-CoV-2 real-time reverse transcriptase polymerase chain reaction during their pregnancy or 14 days preconception and newborns born to mothers infected with SARS-CoV-2 will be included. They will continue to be followed up until 4 weeks after delivery. The aim of the study is to investigate both the effect of COVID-19 on the pregnancy, and the effect of the pregnancy status with the evolution of the SARS-CoV-2 disease. Other samples (faeces, urine, serum, amniotic fluid, cord and peripheral blood, placenta and breastmilk) will be collected in order to analyse whether or not there is a risk of vertical transmission and to describe the behaviour of the virus in other fluids. Neonates will be followed until 6 months after delivery to establish the rate of neonatal transmission. We aim to include 150 pregnant women and their babies. Ethics approval will be obtained from all the participating centres. DISCUSSION: There is little information known about COVID-19 and its unknown effects on pregnancy. This study will collect a large number of samples in pregnant women which will allow us to demonstrate the behaviour of the virus in pregnancy and postpartum in a representative cohort of the Spanish population.

A pilot trial of human amniotic fluid for the treatment of COVID-19.

OBJECTIVE: Vertical transmission from SARS CoV-2-infected women is uncommon and coronavirus has not been detected in amniotic fluid. Human amniotic products have a broad immune-mediating profile. Observing that many COVID-19 patients have a profound inflammatory response to the virus, we sought to determine the influence of human amniotic fluid (hAF) on hospitalized patients with COVID-19. RESULTS: A 10-patient case series was IRB-approved to study the impact of hAF on hospitalized patients with documented COVID-19. Nine of the 10 patients survived to discharge, with one patient succumbing to the disease when enrolled on maximal ventilatory support and severe hypoxia. The study design was altered by the IRB such that the last 6 patients received higher dose of intravenous hAF. In this latter group, patients that had observed reductions in C-reactive protein were associated with improved clinical outcomes. No hAF-related adverse events were noted. Acknowledging some of the inherent limitations of this case series, these results inform and catalyze a larger scaled randomized prospective trial to further investigate hAF as a therapy for COVID-19. Trial Registration ClinicalTrials.gov: NCT04319731; March 23, 2020.

Current Status and Future Prospects of Perinatal Stem Cells.

The placenta is a temporary organ that is discarded after birth and is one of the most promising sources of various cells and tissues for use in regenerative medicine and tissue engineering, both in experimental and clinical settings. The placenta has unique, intrinsic features because it plays many roles during gestation: it is formed by cells from two individuals (mother and fetus), contributes to the development and growth of an allogeneic fetus, and has two independent and interacting circulatory systems. Different stem and progenitor cell types can be isolated from the different perinatal tissues making them particularly interesting candidates for use in cell therapy and regenerative medicine. The primary source of perinatal stem cells is cord blood. Cord blood has been a well-known source of hematopoietic stem/progenitor cells since 1974. Biobanked cord blood has been used to treat different hematological and immunological disorders for over 30 years. Other perinatal tissues that are routinely discarded as medical waste contain non-hematopoietic cells with potential therapeutic value. Indeed, in advanced perinatal cell therapy trials, mesenchymal stromal cells are the most commonly used. Here, we review one by one the different perinatal tissues and the different perinatal stem cells isolated with their phenotypical characteristics and the preclinical uses of these cells in numerous pathologies. An overview of clinical applications of perinatal derived cells is also described with special emphasis on the clinical trials being carried out to treat COVID19 pneumonia. Furthermore, we describe the use of new technologies in the field of perinatal stem cells and the future directions and challenges of this fascinating and rapidly progressing field of perinatal cells and regenerative medicine.

Fetal deaths in pregnancies with SARS-CoV-2 infection in Brazil: A case series.

BACKGROUND: There are few reports of miscarriages or stillbirths in women infected with SARS-CoV-2. We present five consecutive cases of fetal death (≥12 weeks) without other putative causes in women with laboratory-confirmed (RT-PCR) COVID-19 managed in a single Brazilian institution. CASE SERIES: All five women were outpatients with mild or moderate forms of COVID-19 and were not taking any medication. Four were nulliparous, all were overweight or obese, and none had any comorbidities or pregnancy complications that could contribute to fetal demise. Fetal death occurred at 21-38 weeks of gestation, on COVID-days 1-22. SARS-Cov-2 was detected by RT-PCR in amniotic fluid in one case and in placental specimens in two cases. All five women had acute chorioamnionitis on placental histology, massive deposition of fibrin, mixed intervillitis/villitis, and intense neutrophil and lymphocyte infiltration. One fetus had neutrophils inside alveolar spaces, suggestive of fetal infection. CONCLUSIONS: These five cases of fetal demise in women with confirmed COVID-19 without any other significant clinical or obstetric disorders suggest that fetal death can be an outcome of SARS-CoV-2 infection in pregnancy. The intense placental inflammatory reaction in all five cases raises the possibility of a direct effect of SARS-CoV-2 on the placenta.

Does the maternal-fetal transmission of SARS-CoV-2 occur during pregnancy?

BACKGROUND AND OBJETIVE: On January 7th, 2020, a new coronavirus, SARS-CoV-2, was identified, as responsible for a new human disease: COVID-19. Given its recent appearance, our current knowledge about the possible influence that this disease can exert on pregnancy is very limited. One of the unknowns to be solved is whether there is a vertical transmission of the infection during pregnancy. PATIENTS AND METHODS: Using the Real-time Polymerase Chain Reaction techniques for SARS-CoV-2 nucleic acids, the possible presence of this germ in vaginal discharge and amniotic fluid was investigated in four pregnant Caucasian patients affected by mild acute symptoms of COVID-19 during the second trimester of pregnancy. RESULTS: There is no laboratory evidence to suggest a possible passage of SARS-CoV-2 from the infected mother to the amniotic fluid. CONCLUSIONS: It is necessary to expand the investigation of COVID-19 cases diagnosed during pregnancy to clarify the real influence that SARS-CoV-2 has on pregnant women and their offspring, as well as those factors that modulate the disease.