Human Amniotic Fluid Research Articles

The highly packed and dehydrated structure of preformed unexposed human pulmonary surfactant isolated from amniotic fluid.

By coating the alveolar air-liquid interface, lung surfactant overwhelms surface tension forces that, otherwise, would hinder the lifetime effort of breathing. Years of research have provided a picture of how highly hydrophobic and specialized proteins in surfactant promote rapid and efficient formation of phospholipid-based complex three-dimensional films at the respiratory surface, highly stable under the demanding breathing mechanics. However, recent evidence suggests that the structure and performance of surfactant typically isolated from bronchoalveolar lung lavages may be far from that of nascent, still unused, surfactant as freshly secreted by type II pneumocytes into the alveolar airspaces. In the present work, we report the isolation of lung surfactant from human amniotic fluid (amniotic fluid surfactant, AFS) and a detailed description of its composition, structure, and surface activity in comparison to a natural surfactant (NS) purified from porcine bronchoalveolar lavages. We observe that the lipid/protein complexes in AFS exhibit a substantially higher lipid packing and dehydration than in NS. AFS shows melting transitions at higher temperatures than NS and a conspicuous presence of nonlamellar phases. The surface activity of AFS is not only comparable with that of NS under physiologically meaningful conditions but displays significantly higher resistance to inhibition by serum or meconium, agents that inactivate surfactant in the context of severe respiratory pathologies. We propose that AFS may be the optimal model to study the molecular mechanisms sustaining pulmonary surfactant performance in health and disease, and the reference material to develop improved therapeutic surfactant preparations to treat yet unresolved respiratory pathologies.

Human amniotic fluid derived-exosomes alleviate hypoxic encephalopathy by enhancing angiogenesis in neonatal mice after hypoxia

Neonatal hypoxic encephalopathy is a type of central nervous system dysfunction manifested by high mortality and morbidity. Exosomes play a crucial role in neuroprotection by enhancing angiogenesis. The objective of this study was to investigate the effect of human amniotic fluid-derived exosomes (hAFEXOs) on functional recovery in neonatal hypoxic encephalopathy. The transwell assay, scratch wound healing assay, and tube formation assay were used to evaluate the effect of hAFEXOs on the angiogenesis of human umbilical vein endothelial cells (HUVECs) after oxygen and glucose deprivation (OGD). The angiogenesis of microvascular endothelial cells (MECs) in the cortex was tested in neonatal mice treated with hAFEXOs or phosphate-buffered saline (PBS) after hypoxia. Expressions of hypoxia-inducible factor 1 α (HIF-1α) and vascular endothelial growth factor (VEGF) in the cerebral cortex were also tested by western blot. The Morris Water Maze Test (MWM) was carried out to detect the performance of spatial memory after processing with hAFEXOs or PBS. The results indicated that hAFEXOs favored tubing formation and migration of HUVECs after in vitro OGD. The hAFEXOs also favored the expression of CD31 in neonatal mice following hypoxia. The expressions of both HIF-1α and VEGF were significantly augmented in the cerebral cortex of neonatal mice which were treated with hAFEXOs. Moreover, the MWM test results showed that the performance of the spatial memory was better in the hAFEXO-treated group than in the PBS-treated group. Our study indicates that hAFEXOs alleviated hypoxic encephalopathy and enhanced angiogenesis in neonatal mice after hypoxia. In addition, hAFEXOs promoted migration and tube formation of HUVECs after OGD in vitro. These findings confirm that hAFEXOs show great potential for further studies aimed at developing therapeutic agents for hypoxic encephalopathy.

Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery

Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is in utero delivery of messenger RNA (mRNA). mRNA is a nucleic acid therapeutic that has previously been investigated as a platform for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to risks associated with immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects their stability in amniotic fluid ex utero and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in primary fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery.

Human Amniotic Fluid for the Treatment of Hospitalized, Symptomatic, and Laboratory-verified SARS-CoV-2 Patients

New reports offer evidence that under different circumstances, intrauterine mother-infant transmission of SARS-CoV-2 occurs. In contrast, early observations in the COVID-19 pandemic recommended that vertical transmission from women infected with SARS-CoV-2 can be challenging and no virus is detected in human amniotic fluid (HAF). The present study aimed to propose the idea that HAF can be used as a potential therapy for hospitalized, symptomatic, and laboratory-verified SARS-CoV-2 patients by mitigating COVID-19 related inflammation and decreasing its fibrosis. Considering that COVID-19 can cause a severe pulmonary fibrotic response in some patients, HAF by decreasing fibrosis may be considered as an alternative and novel therapy against COVID-19. Lastly, given the inexpensive, easy to access, and safe nature of HAF, integrating this therapy may decrease the COVID-19 attributed death and burden to the health system, especially in countries with limited access to vaccines where HAF is widely available.

The Role of Innate Immune System in the Human Amniotic Membrane and Human Amniotic Fluid in Protection Against Intra-Amniotic Infections and Inflammation

Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI.

Morphological study of equine amniotic compartment

Exfoliative cytology of human amniotic fluid (AF) has been extensively studied since 1940s, but no data exist in equine species. The AF compartment represents the environment in which the foetus grows and matures, and its composition changes, reflecting foetal well-being and development. The aim of this study was to describe for the first time the morphology of equine AF cells and amniotic membrane (AM) with light microscopy (LM) and transmission electron microscopy (TEM). AF was collected at parturition within 5 min after the appearance of the AM with a 60 mL syringe from 34 mares and samples of AM were collected from a subset of 7 mares with normal pregnancy hospitalized for attended parturition. For LM observation, a sample of cytocentrifuged fresh AF was stained with May-Grünwald Giemsa and AM sections were stained with H-E. For TEM observation, AF and AM were fixed, embedded in epoxy resins, then sectioned and stained with uranyl acetate and lead citrate solutions. Nucleated and anucleated squamous cells with basophilic cytoplasm, intensely basophilic cornified cells, polymorphonuclear cells, and clusters of eosinophilic amorphous substance were observed. Cells presumably derived from tracheal epithelium and small round nucleated cells with eosinophilic cytoplasm presumably derived from amniotic or urinary epithelium were occasionally found. Lamellar body-like structures (LBs) were present in some epithelial cells. In AM, epithelial, basal and mesenchymal layers were clearly visible with both techniques as previously described. Epithelial cells had several cytoplasmic vacuolization and microvilli were present on apical surface. The connective tissue presented fibroblasts, mesenchymal and rare polymorphonuclear cells, surrounded by abundant extracellular matrix, with distribution of collagen fibres. This is the first report about equine amniotic compartment description by LM and TEM. As recently reported in human medicine, the AM could be a second potential source of pulmonary surfactant, given the finding of LBs inside the cells which could have the same function as in humans. Further studies in samples collected at different gestational ages could increase the knowledge of AF cells and their modification during pregnancy, as well as a better comprehension of the role of AM as a secondary source of pulmonary surfactant in the horse. The diagnostic evaluation of AF cellular composition in high-risk pregnancies may also be investigated.

A Prevascularized Polyurethane-Reinforced Fibrin Patch Improves Regenerative Remodeling in a Rat Right Ventricle Replacement Model.

Congenital heart defects (CHDs) affect 1 in 120 newborns in the United States. Surgical repair of structural heart defects often leads to arrhythmia and increased risk of heart failure. The laboratory has previously developed an acellular fibrin patch reinforced with a biodegradable poly(ether ester urethane) urea mesh that result in improved heart function when tested in a rat right ventricle wall replacement model compared to fixed pericardium. However, this patch does not drive significant neotissue formation. The patch materials are modified here and this patch is prevascularized with human umbilical vein endothelial cells and c-Kit+ human amniotic fluid stem cells. Rudimentary capillary-like networks form in the fibrin after culture of cell-encapsulated patches for 3 d in vitro. Prevascularized patches and noncell loaded patch controls are implanted onto full-thickness heart wall defects created in the right ventricle of athymic nude rats. Two months after surgery, defect repair with prevascularized patches results in improved heart function and the patched heart area exhibited greater vascularization and muscularization, less fibrosis, and increased M2 macrophage infiltration compared to acellular patches.

Human amniotic fluid stem cells can improve cerebral vascular remodelling and neurological function after focal cerebral ischaemia in diabetic rats

Diabetes causes vascular injury and carries a high risk of ischaemic stroke. Human amniotic fluid stem cells (hAFSCs) can enhance cerebral vascular remodelling and have the potential to improve neurological function after stroke in diabetic rats. Five groups of female rats were examined: (1) normal control, (2) type 1 diabetic (T1DM) rats induced by streptozotocin injection (DM), (3) non‐DM rats receiving 60‐minute middle cerebral artery occlusion (MCAO), (4) T1DM rats receiving 60‐minute MCAO (DM + MCAO) and (5) T1DM rats receiving 60‐minute MCAO and injection with 5 × 106 hAFSCs at 3 h after MCAO (DM + MCAO + hAFSCs). Neurological function was examined before, and at 1, 7, 14, 21 and 28 days, and cerebral infarction volume and haemorrhage, cerebral vascular density, angiogenesis and inflammatory were examined at 7 and 28 days after MCAO. hAFSCs treatment caused a significant improvement of neurological dysfunction, infarction volume, blood‐brain barrier leakage, cerebral arterial density, vascular density and angiogenesis and a reduction of brain haemorrhage and inflammation compared with non‐treatment. Our results showed that the effect of hAFSCs treatment against focal cerebral ischaemia may act through the recovery of vascular remodelling and angiogenesis and the reduction of inflammation in ischaemic brain.

Identification of known and novel nonpolar endocrine disruptors in human amniotic fluid

BackgroundPrenatal exposure to endocrine-disrupting compounds (EDCs) may contribute to endocrine-related diseases and disorders later in life. Nevertheless, data on in utero exposure to these compounds are still scarce. ObjectivesWe investigated a wide range of known and novel nonpolar EDCs in full-term human amniotic fluid (AF), a representative matrix of direct fetal exposure. MethodsGas chromatography high-resolution mass spectrometry (GC-HRMS) was used for the targeted and non-targeted analysis of chemicals present in nonpolar AF fractions with dioxin-like, (anti-)androgenic, and (anti-)estrogenic activity. The contribution of detected EDCs to the observed activity was determined based on their relative potencies. The multitude of features detected by non-targeted analysis was tentatively identified through spectra matching and data filtering, and further investigated using curated and freely available sources to predict endocrine activity. Prioritized suspects were purchased and their presence in AF was chemically and biologically confirmed with GC-HRMS and bioassay analysis. ResultsTargeted analysis revealed 42 known EDCs in AF including dioxins and furans, polybrominated diphenyl ethers, pesticides, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons. Only 30% of dioxin activity and <1% estrogenic and (anti-)androgenic activity was explained by the detected compounds. Non-targeted analysis revealed 14,110 features of which 3,243 matched with library spectra. Our data filtering strategy tentatively identified 121 compounds. Further data mining and in silico predictions revealed in total 69 suspected EDCs. We selected 14 chemicals for confirmation, of which 12 were biologically active and 9 were chemically confirmed in AF, including the plasticizer diphenyl isophthalate and industrial chemical p,p'-ditolylamine. ConclusionsThis study reveals the presence of a wide variety of nonpolar EDCs in direct fetal environment and for the first time identifies novel EDCs in human AF. Further assessment of the source and extent of human fetal exposure to these compounds is warranted.

The Restorative Effect of Human Amniotic Fluid Stem Cells on Spinal Cord Injury.

Spinal cord injury (SCI) is a debilitating condition within the neural system which is clinically manifested by sensory-motor dysfunction, leading, in some cases, to neural paralysis for the rest of the patient’s life. In the current study, mesenchymal stem cells (MSCs) were isolated from the human amniotic fluid, in order to study their juxtacrine and paracrine activities. Flow cytometry analysis was performed to identify the MSCs. A conditioned medium (CM) was collected to measure the level of BDNF, IL-1β, and IL-6 proteins using the ELISA assay. Following the SCI induction, MSCs and CM were injected into the lesion site, and also CM was infused intraperitoneally in the different groups. Two weeks after SCI induction, the spinal cord samples were examined to evaluate the expression of the doublecortin (DCX) and glial fibrillary acid protein (GFAP) markers using immunofluorescence staining. The MSCs’ phenotype was confirmed upon the expression and un-expression of the related CD markers. Our results show that MSCs increased the expression level of the DCX and decreased the level of the GFAP relative to the injury group (p < 0.001). Additionally, the CM promoted the DCX expression rate (p < 0.001) and decreased the GFAP expression rate (p < 0.01) as compared with the injury group. Noteworthily, the restorative potential of the MSCs was higher than that of the CM (p < 0.01). Large-scale meta-analysis of transcriptomic data highlighted PAK5, ST8SIA3, and NRXN1 as positively coexpressed genes with DCX. These genes are involved in neuroactive ligand–receptor interaction. Overall, our data revealed that both therapeutic interventions could promote the regeneration and restoration of the damaged neural tissue by increasing the rate of neuroblasts and decreasing the astrocytes.

Non-Nutritive Sweeteners in Human Amniotic Fluid and Cord Blood: Evidence of Transplacental Fetal Exposure.

This study aimed to investigate human fetal exposure to non-nutritive sweeteners (NNS) by analyzing amniotic fluid and umbilical cord blood. Concentrations of four NNS (acesulfame-potassium [ace-K], saccharin, steviol glucuronide, and sucralose) were measured in amniotic fluid (n = 13) and cord blood samples (n = 15) using liquid chromatography-mass spectrometry. Amniotic fluid samples were obtained for research purposes at the time of term elective cesarean birth or clinically indicated third trimester amnioreduction at Mercy Hospital for Women (Melbourne, Australia). All except four women were in the fasting state. Cord blood samples were obtained from an independent cohort of newborns whose mothers were enrolled in a separate clinical trial at the National Institutes of Health. Ten of 13 amniotic fluid samples contained at least one NNS (ace-K, saccharin, steviol glucuronide, and/or sucralose). Maximum amniotic fluid NNS concentrations of ace-K, saccharin, steviol glucuronide, and sucralose were 78.9, 55.9, 93.5, and 30.6 ng/mL, respectively. Ace-K and saccharin were present in 100% and 80% of the cord blood samples, with maximal concentrations of 6.5 and 2.7 ng/mL, respectively. Sucralose was not detected and steviol glucuronide was not measurable in any of the cord blood samples. Our results provide evidence of human transplacental transmission of NNS. Based on results predominantly obtained from rodent models, we speculate that NNS exposure may adversely influence the offsprings' metabolic health. Well-designed, prospective clinical trials are necessary to understand the impact of NNS intake during pregnancy on human development and long-term health. · NNS consumption during pregnancy has increased in recent years.. · Maternal NNS intake during pregnancy is associated with preterm birth and higher infant weight gain in epidemiologic studies.. · In rodents, in utero NNS exposure induces metabolic abnormalities in mothers and their offspring, alters offspring gut microbiota composition, and promotes sweet taste preference in adulthood.. · It is presently unknown whether and to what degree maternal NNS ingestion in humans leads to direct in utero exposure.. · This study provides the first evidence of in utero NNS exposure in humans and highlights the urgent need to investigate clinical consequences of early life NNS exposure on metabolism, weight, taste preference, and general health..

Three-dimensional migration of human amniotic fluid stem cells involves mesenchymal and amoeboid modes and is regulated by mTORC1.

Three‐dimensional (3D) cell migration is an integral part of many physiologic processes. Although being well studied in the context of adult tissue homeostasis and cancer development, remarkably little is known about the invasive behavior of human stem cells. Using two different kinds of invasion assays, this study aimed at investigating and characterizing the 3D migratory capacity of human amniotic fluid stem cells (hAFSCs), a well‐established fetal stem cell type. Eight hAFSC lines were found to harbor pronounced potential to penetrate basement membrane (BM)‐like matrices. Morphological examination and inhibitor approaches revealed that 3D migration of hAFSCs involves both the matrix metalloprotease‐dependent mesenchymal, elongated mode and the Rho‐associated protein kinase‐dependent amoeboid, round mode. Moreover, hAFSCs could be shown to harbor transendothelial migration capacity and to exhibit a motility‐associated marker expression pattern. Finally, the potential to cross extracellular matrix was found to be induced by mTORC1‐activating growth factors and reduced by blocking mTORC1 activity. Taken together, this report provides the first demonstration that human stem cells exhibit mTORC1‐dependent invasive capacity and can concurrently make use of mesenchymal and amoeboid 3D cell migration modes, which represents an important step toward the full biological characterization of fetal human stem cells with relevance to both developmental research and stem cell‐based therapy.

Amphiregulin stimulates human chorionic gonadotropin expression by inducing ERK1/2-mediated ID3 expression in trophoblast cells

IntroductionThe human chorionic gonadotropin (hCG) is a dimer consisting of an α subunit and a β subunit which is encoded by the CGB gene and is unique to hCG. hCG is a hormone mainly synthesized by syncytiotrophoblast cells in the placenta, plays a critical role in stimulating progesterone production that is necessary for maintaining normal pregnancy in the early stage. Epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinase family which has been shown to regulate various physiological and pathological events. In human chorionic villi and amniotic fluid, amphiregulin (AREG) is reported to be the most abundant EGFR ligand and can stimulate hCG expression. However, the underlying mechanism remains unknown. MethodsWe use BeWo cells, the commonly used cell model for the hCG production of trophoblast cells, as an in vitro model. The effects of AREG on CGB expression and hCG secretion as well as the underlying mechanisms were explored by a series of in vitro experiments. ResultsWe show that treatment with AREG stimulates CGB expression and hCG secretion. Using pharmacological inhibitors, we show that the stimulatory effects of AREG on CGB expression and hCG secretion are mediated by the EGFR-activated ERK1/2 signaling pathways. In addition, the expression of inhibitor of DNA-binding protein 3 (ID3) is upregulated by AREG. Knockdown of ID3 attenuates the AREG-induced upregulation of CGB expression and hCG secretion. DiscussionThis study provides important insights into the molecular mechanisms that mediate AREG-induced upregulation of hCG production in human trophoblast cells which may lead to the development of alternative therapeutic approaches for the treatment of placental diseases.

Metabolic Profile and Neurogenic Potential of Human Amniotic Fluid Stem Cells From Normal vs. Fetus-Affected Gestations.

Human amniotic fluid stem cells (hAFSCs) possess some characteristics with mesenchymal stem cells (MSCs) and embryonic stem cells and have a broader differentiation potential compared to MSCs derived from other sources. Although hAFSCs are widely researched, their analysis mainly involves stem cells (SCs) obtained from normal, fetus-unaffected gestations. However, in clinical settings, knowledge about hAFSCs from normal gestations could be poorly translational, as hAFSCs from healthy and fetus-diseased gestations may differ in their differentiation and metabolic potential. Therefore, a more thorough investigation of hAFSCs derived from pathological gestations would provide researchers with the knowledge about the general characteristics of these cells that could be valuable for further scientific investigations and possible future clinical applicability. The goal of this study was to look into the neurogenic and metabolic potential of hAFSCs derived from diseased fetuses, when gestations were concomitant with polyhydramnios and compare them to hAFSCs derived from normal fetuses. Results demonstrated that these cells are similar in gene expression levels of stemness markers (SOX2, NANOG, LIN28A, etc.). However, they differ in expression of CD13, CD73, CD90, and CD105, as flow cytometry analysis revealed higher expression in hAFSCs from unaffected gestations. Furthermore, hAFSCs from “Normal” and “Pathology” groups were different in oxidative phosphorylation rate, as well as level of ATP and reactive oxygen species production. Although the secretion of neurotrophic factors BDNF and VEGF was of comparable degree, as evaluated with enzyme-linked immunosorbent assay (ELISA) test, hAFSCs from normal gestations were found to be more prone to neurogenic differentiation, compared to hAFSCs from polyhydramnios. Furthermore, hAFSCs from polyhydramnios were distinguished by higher secretion of pro-inflammatory cytokine TNFα, which was significantly downregulated in differentiated cells. Overall, these observations show that hAFSCs from pathological gestations with polyhydramnios differ in metabolic and inflammatory status and also possess lower neurogenic potential compared to hAFSCs from normal gestations. Therefore, further in vitro and in vivo studies are necessary to dissect the potential of hAFSCs from polyhydramnios in stem cell-based therapies. Future studies should also search for strategies that could improve the characteristics of hAFSCs derived from diseased fetuses in order for those cells to be successfully applied for regenerative medicine purposes.

Evidence of bacterial DNA presence in chorionic villi and amniotic fluid in the first and second trimester of pregnancy.

The sterile-womb dogma in uncomplicated pregnancy has been lively debated. Data regarding the in utero microbiome environment are based mainly on studies performed at the time of delivery. Aim: To determine whether human placenta and amniotic fluid are populated by a bacterial microbiota in the first and second trimesters of pregnancy. Materials & methods: We analyzed by next-generation sequencing method 24 and 29 samples from chorionic villus sampling (CVS) and amniocentesis (AC), respectively. The V3 region of the 16S rRNA gene was sequenced. Results: 37.5% of CVS and 14% of AC samples showed the presence of bacterial DNA. Conclusion: Our study suggests that bacterial DNA can be identified in the placenta and amniotic fluid during early prenatal life.

Human Amniotic Fluid Stem Cell-Derived Exosomes as a Novel Cell-Free Therapy for Cutaneous Regeneration.

Adult wound healing often results in fibrotic scarring that is caused by myofibroblast aggregation. Human amniotic fluid stem cells (hAFSCs) exhibit significantly anti-fibrotic scarring properties during wound healing. However, it is little known whether hAFSCs directly or indirectly (paracrine) contribute to this process. Using the full-thickness skin-wounded rats, we investigated the therapeutic potential of hAFSC-derived exosomes (hAFSC-exo). Our results showed that hAFSC-exo accelerated the wound healing rate and improved the regeneration of hair follicles, nerves, and vessels, as well as increased proliferation of cutaneous cells and the natural distribution of collagen during wound healing. Additionally, hAFSC-exo suppressed the excessive aggregation of myofibroblasts and the extracellular matrix. We identified several miRNAs, including let-7-5p, miR-22-3p, miR-27a-3p, miR-21-5p, and miR-23a-3p, that were presented in hAFSC-exo. The functional analysis demonstrated that these hAFSC-exo-miRNAs contribute to the inhibition of the transforming growth factor-β (TGF-β) signaling pathway by targeting the TGF-β receptor type I (TGF-βR1) and TGF-β receptor type II (TGF-βR2). The reduction of TGF-βR1 and TGF-βR2 expression induced by hAFSC-exo was also confirmed in the healing tissue. Finally, using mimics of miRNAs, we found that hAFSC-exo-miRNAs were essential for myofibroblast suppression during the TGF-β1-induced human dermal fibroblast-to-myofibroblast transition in vitro. In summary, this study is the first to show that exosomal miRNAs used in hAFSC-based therapy inhibit myofibroblast differentiation. Our study suggests that hAFSC-exo may represent a strategic tool for suppressing fibrotic scarring during wound healing.

The Presence of Mycotoxins in Human Amniotic Fluid.

Mycotoxin exposure assessments through biomonitoring studies, based on the analysis of amniotic fluid, provides useful information about potential exposure of mothers and fetuses to ubiquitous toxic metabolites that are routinely found in food and the environment. In this study, amniotic fluid samples (n = 86) were collected via abdominal amniocentesis at 15–22 weeks of gestation from pregnant women with a high risk of chromosomal anomalies or genetic fetal defects detected during 1st trimester prenatal screening. These samples were analyzed for the presence of the most typical Aspergillus, Penicillium and Fusarium mycotoxins, with a focus on aflatoxins, ochratoxins and trichothecenes, using the LC-FLD/DAD method. The results showed that the toxin was present in over 75% of all the tested samples and in 73% of amniotic fluid samples from fetuses with genetic defects. The most frequently identified toxins were nivalenol (33.7%) ranging from <LOQ to 4037.6 ng/mL, and aflatoxins (31.4%), including aflatoxin G1, ranging from <LOQ to 0.4 ng/mL. Ochratoxin A and deoxynivalenol were identified in 26.7% and 27.9% of samples, respectively. Bearing in mind the above, the detection of mycotoxin levels in amniotic fluid is useful for the estimation of overall risk characterization with an attempt to link the occurrence of fetal abnormalities with exposure to mycotoxins in utero.

83 Dehydrated human amniotic membrane and amniotic fluid for the treatment of rectovaginal fistulas

The presence of a rectovaginal fistula (RVF) can be devastating to patients thus greatly affecting their quality of life. The most common etiologies of RVF are those resulting from obstetric complications, pelvic radiation, malignancy, infection, and postoperative complications. The treatment of RVF is mainly surgical; however, current techniques still have a high rate of recurrence. Our objective is to present a novel surgical technique with the use of a dehydrated amniotic membrane (VendajeTM) and an amniotic suspension allograft (RheoTM) in repair of rectovaginal fistula. Two cases of chronic rectovaginal fistula were seen in the urogynecology office. Informed consent was obtained to proceed with surgical repair using the dehydrated amniotic membrane and amniotic suspension allograft. Under general anesthesia, the RVF fistula repair was done by first using a #5 silk suture that was threaded back and forth in the fistula tract to de-epithelialize the tract and uncover healthy tissue (Figure 1). The rectal side of the fistula tract was closed with 3-0 vicryl interrupted sutures. The amniotic membrane was soaked in 1 mL of the amniotic suspension before being placed into the fistula tract from the vaginal side. The vaginal side of the tract was reapproximated with 3-0 vicryl interrupted sutures (Figure 2). Finally, the remainder of the amniotic suspension was injected into the tract. The patients were also advised to use estrace vaginal cream to promote wound healing and increase blood supply to the surgical site. One case of complete resolution (5 mm to closure) of rectovaginal fistula and one case of 90% improvement (1cm to 1mm) of fistula is presented utilizing a novel therapeutic approach with the amniotic membrane pre-soaked in an amniotic suspension allograft followed by injection of remaining allograft into the fistula tract. This novel application of dehydrated human amniotic membrane allograft and amniotic suspension allograft is an effective therapy for chronic rectovaginal fistula. This procedure also highlights the importance of the complexity of the fistula and health of the supporting tissue prior to fistula repair seen in the case with 70% improvement.

Stem cells from the amniotic fluid: a promising tool to face the cytokine storm associated to SARS‐CoV‐2 infections

Infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the etiological agent of coronavirus disease 19 (COVID‐19) is associated with significant morbidity and mortality. Despite significant progress since the emergence of this disease, efficacious therapeutics are still urgently required. Infection is associated with a broad spectrum of symptoms and varying disease severity. In severe cases, a multi‐organ disease is apparent, involving the respiratory, coagulation, immune and cardiac systems. A central tenant of severe disease is an excessive immune response to infection leading to a cytokine storm and associated organ and tissue damage. Immunological therapies capable of attenuating the cytokine storm may therefore provide novel treatment options, and the present study explored whether mesenchymal stem cells (MSCs) from the amniotic fluid (AF) and their derivatives, through their immunomodulatory, anti‐oxidant, and regenerative functions, could provide such an option.Next Generation Sequencing analysis of MSCs isolated from human AF identified a plethora of molecules with the potential to positively influence the prognosis in SARS‐Cov‐2 infection patients. These included mRNAs involved in the regulation of several immune pathways that are basis of the impaired immune response and cytokine storm caused seen in SARS‐CoV‐2‐positive patients, such as: HIF‐1, IL‐17, Toll‐like receptors, RAP1, TNF, WNT, PI3K‐Akt and NF‐kappa B signalling. AF‐MSCs also contained bioactive molecules involved in respiratory endothelial protection and repair, such as VEGF, IL‐1, TGF‐β1, EGFR, in extracellular matrix re‐organization or associated with cardiac muscle cell function. Analysis of non‐coding RNAs identified the presence of microRNAs that regulate macrophage polarization (towards an anti‐inflammatory phenotype) and attenuate inflammation, as well as regulate the host response to viral infection. Among those, exosomal miRNAs have been also identified.Building upon these observations, further efforts will be made to elucidate the potential therapeutic use of AF‐MSCs and their derivatives in combating the immune‐related side effects of SARS‐CoV‐2. The successful incorporation of such an MSC‐based therapy into the treatment regimen of SARS‐CoV‐2 infected patients has significant potential to reduce the morbidity and mortality associated with COVID‐19.

Prenatal transplantation of human amniotic fluid stem cell could improve clinical outcome of type III spinal muscular atrophy in mice

Spinal muscular atrophy (SMA) is a single gene disorder affecting motor function in uterus. Amniotic fluid is an alternative source of stem cell to ameliorate SMA. Therefore, this study aims to examine the therapeutic potential of Human amniotic fluid stem cell (hAFSC) for SMA. Our SMA model mice were generated by deletion of exon 7 of Smn gene and knock-in of human SMN2. A total of 16 SMA model mice were injected with 1 × 105 hAFSC in uterus, and the other 16 mice served as the negative control. Motor function was analyzed by three behavioral tests. Engraftment of hAFSC in organs were assessed by flow cytometry and RNA scope. Frequency of myocytes, neurons and innervated receptors were estimated by staining. With hAFSC transplantation, 15 fetuses survived (93.75% survival) and showed better performance in all motor function tests. Higher engraftment frequency were observed in muscle and liver. Besides, the muscle with hAFSC transplantation expressed much laminin α and PAX-7. Significantly higher frequency of myocytes, neurons and innervated receptors were observed. In our study, hAFSC engrafted on neuromuscular organs and improved cellular and behavioral outcomes of SMA model mice. This fetal therapy could preserve the time window and treat in the uterus.