Ferroptosis induces membrane blebbing in placental trophoblasts.

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.

PLA2G6 guards placental trophoblasts against ferroptotic injury.

The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.

Safety assessment of the prophylactic use of silicone gel sheets (Lady Care®) for the prevention of hypertrophic scars following caesarean section.

This study aimed to investigate the side effects of silicone gel sheet (Lady Care®) and evaluate its prophylactic efficacy in preventing abnormal scarring. Sixty women who underwent caesarean section were recruited from September 2016 to September 2017 in this prospective study. Lady Care® was applied from the 2nd to the 6th postoperative months. Side effects of Lady Care® were evaluated through medical examinations and questionnaires. A plastic surgeon diagnosed abnormal scarring. Pruritus was diagnosed in 25 (47.2%) patients; folliculitis, four (7.5%); dry skin, four (7.5%); contact dermatitis, three (5.7%); wound infection, two (3.8%); and epidermolysis, one (1.9%), albeit with mild severity. Following Lady Care® application, no abnormal scarring and mild hypertrophic scarring was observed in 32 (64.0%) and 18 (36.0%) patients respectively. Of seven patients with pre-existing hypertrophic scars, only two showed hypertrophic scarring after Lady Care® application. Our findings support the safety and prophylactic efficacy of Lady Care®.Impact StatementWhat is already known on this subject? The incidence of abnormal scarring, i.e. keloid or hypertrophic scar formation after caesarean section (CS) is reported to be ∼41%. Abnormal or excessive scar formation can lead to functional limitations, pruritus, pain and cosmetic issues. Studies have also shown a prophylactic effect of the application of silicone materials against the development of hypertrophic and keloid scars, though prohibitive cost and lack of adhesiveness of such gel sheets are known factors limiting their usage.What the results of this study add? The new silicone gel sheet 'Lady Care®' has strong adhesive properties and is consequently not easily peeled off. Furthermore, it is easy to use and economically efficient.What the implications are of these findings for clinical practice and/or further research? This is the first clinical trial on the application of Lady Care® silicone gel sheet for the prevention of CS scarring. Our findings support the safety and prophylactic efficacy of Lady Care®.

Autonomous trisomic rescue of Down syndrome cells.

Down syndrome is the most frequent chromosomal abnormality among live-born infants. All Down syndrome patients have mental retardation and are prone to develop early onset Alzheimer's disease. However, it has not yet been elucidated whether there is a correlation between the phenotype of Down syndrome and the extra chromosome 21. In this study, we continuously cultivated induced pluripotent stem cells (iPSCs) with chromosome 21 trisomy for more than 70 weeks, and serendipitously obtained revertant cells with normal chromosome 21 diploids from the trisomic cells during long-term cultivation. Repeated experiments revealed that this trisomy rescue was not due to mosaicism of chromosome 21 diploid cells and occurred at an extremely high frequency. We herewith report the spontaneous correction from chromosome 21 trisomy to disomy without genetic manipulation, chemical treatment or exposure to irradiation. The revertant diploid cells will possibly serve a reference for drug screening and a raw material of regenerative medicinal products for cell-based therapy.

Clinical experience of J-VAC drain for skin closure in the laparotomy of obstetrics and gynecology.

AIM: The frequency of wound dehiscence after abdominal surgery has been reported to be approximately 4-29%, and that of surgical site infections is said to be of about 20%. We examined the effectiveness of the subcutaneous J-VAC drain (JVD) in the drainage of bleeding and exudates from surgical wounds. MATERIAL AND METHODS: The study was conducted on 192 patients who underwent abdominal surgery from October 2009 to February 2011, and in whom indwelling JVD were placed. During the study period, JVD (10-Fr) were placed subcutaneously on the anterior surface of the fascia in all patients. We examined the frequency of surgical wound complications. RESULTS: A longitudinal incision was used in 101 patients, and a transverse abdominal incision was used in 91 patients. Subjects with a subcutaneous fat thickness of 2 cm or thicker accounted for 115 patients. Subcutaneous hematoma was present in three patients, but only two patients (1%) showed dehiscence that required treatment. CONCLUSIONS: This study revealed that subcutaneous JVD is useful for the closure of surgical incisions in gynecology and obstetrics, and that there are no limitations to their applicability.

Inhibitory effect of all-trans retinoic acid on ferroptosis in BeWo cells mediated by the upregulation of heme Oxygenase-1.

INTRODUCTION: This study aimed to explore the association between ferroptosis, a newly identified type of cell death, and the role of retinoic acid in developing pregnancy complications. Therefore, the effects of all-trans retinoic acid (ATRA) on ferroptosis susceptibility in BeWo cells were assessed to understand abnormal placental development. METHODS: BeWo cells were used as surrogates for cytotrophoblasts. The effect of ATRA on ferroptosis sensitivity was assessed on BeWo cells pretreated with ATRA or dimethyl sulfoxide (DMSO; control), following which the LDH-releasing assay was performed. The effects of ATRA pretreatment on the antioxidant defense system (including glutathione [GSH], mitochondrial membrane potential, and heme oxygenase-1 [HMOX1]) in BeWo cells were assessed using assay kits, RT-qPCR, and HMOX1 immunostaining. To evaluate the effect of ATRA on BeWo cells, HMOX1 was silenced in BeWo cells using shRNA. RESULTS: ATRA pretreatment increased ferroptosis resistance in BeWo cells. Although with pretreatment, qPCR indicated upregulation of HMOX1, no significant change was observed in the GSH levels or mitochondrial membrane potential. This was corroborated by intensified immunostaining for heme oxygenase-1 protein (HO-1). Notably, the protective effect of ATRA against ferroptosis was negated when HO-1 was inhibited. Although HMOX1-silenced BeWo cells exhibited heightened ferroptosis sensitivity compared with controls, ATRA pretreatment counteracted ferroptosis in these cells. DISCUSSION: ATRA pretreatment promotes BeWo cell viability by suppressing ferroptosis and upregulating HMOX1 and this can be used as a potential therapeutic strategy for addressing placental complications associated with ferroptosis.

Molecular Mechanisms Underlying Twin-to-Twin Transfusion Syndrome.

Twin-to-twin transfusion syndrome is a unique disease and a serious complication occurring in 10-15% of monochorionic multiple pregnancies with various placental complications, including hypoxia, anemia, increased oxidative stress, and ischemia-reperfusion injury. Fetoscopic laser photocoagulation, a minimally invasive surgical procedure, seals the placental vascular anastomoses between twins and dramatically improves the survival rates in twin-to-twin transfusion syndrome. However, fetal demise still occurs, suggesting the presence of causes other than placental vascular anastomoses. Placental insufficiency is considered as the main cause of fetal demise in such cases; however, little is known about its underlying molecular mechanisms. Indeed, the further association of the pathogenic mechanisms involved in twin-to-twin transfusion syndrome placenta with several molecules and pathways, such as vascular endothelial growth factor and the renin-angiotensin system, makes it difficult to understand the underlying pathological conditions. Currently, there are no effective strategies focusing on these mechanisms in clinical practice. Certain types of cell death due to oxidative stress might be occurring in the placenta, and elucidation of the molecular mechanism underlying this cell death can help manage and prevent it. This review reports on the molecular mechanisms underlying the development of twin-to-twin transfusion syndrome for effective management and prevention of fetal demise after fetoscopic laser photocoagulation.

Ferroptosis, trophoblast lipotoxic damage, and adverse pregnancy outcome.

Programmed cell death is a central process in the control of tissue development, organismal physiology, and disease. Ferroptosis is a recently identified form of programmed cell death that is uniquely defined by redox-active iron-dependent hydroxy-peroxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids and a loss of lipid peroxidation repair capacity. This distinctive form of lipotoxic cell death has been recently implicated in multiple human diseases, spanning ischemia-reperfusion heart injury, brain damage, acute kidney injury, cancer, and asthma. Intriguingly, settings that have been associated with ferroptosis are linked to placental physiology and trophoblast injury. Such circumstances include hypoxia-reperfusion during placental development, physiological uterine contractions or pathological changes in placental bed perfusion, the abundance of trophoblastic iron, evidence for lipotoxicity during the pathophysiology of major placental disorders such as preeclampsia, fetal growth restriction, and preterm birth, and reduced glutathione peroxidation capacity and lipid peroxidation repair during placental injury. We recently interrogated placental ferroptosis in placental dysfunction in human and mouse pregnancy, dissected its relevance to placental injury, and validated the role of glutathione peroxidase-4 in guarding placental trophoblasts against ferroptotic injury. We also uncovered a role for the phospholipase PLA2G6 (PNPLA9) in attenuating trophoblast ferroptosis. Here, we summarize current data on trophoblast ferroptosis, and the role of several proteins and microRNAs as regulators of this process. Our text offers insights into new opportunities for regulating ferroptosis as a means for protecting placental trophoblasts against lipotoxic injury.

Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells.

BACKGROUND: An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs). METHODS: Our protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21- and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium. RESULTS: Trisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component. CONCLUSIONS: Keratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions.

Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies.

Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for "endocytic escape" of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.

Gorlin syndrome-induced pluripotent stem cells form medulloblastoma with loss of heterozygosity in PTCH1.

Gorlin syndrome is a rare autosomal dominant hereditary disease with a high incidence of tumors such as basal cell carcinoma and medulloblastoma. Disease-specific induced pluripotent stem cells (iPSCs) and an animal model have been used to analyze disease pathogenesis. In this study, we generated iPSCs derived from fibroblasts of four patients with Gorlin syndrome (Gln-iPSCs) with heterozygous mutations of the PTCH1 gene. Gln-iPSCs from the four patients developed into medulloblastoma, a manifestation of Gorlin syndrome, in 100% (four out of four), of teratomas after implantation into immunodeficient mice, but none (0/584) of the other iPSC-teratomas did so. One of the medulloblastomas showed loss of heterozygosity in the PTCH1 gene while the benign teratoma, i.e. the non-medulloblastoma portion, did not, indicating a close clinical correlation between tumorigenesis in Gorlin syndrome patients and Gln-iPSCs.

Spontaneous Remission of Sick Sinus Syndrome in a Fetus with Pulmonary Stenosis Regurgitation.

Objective Here, we report a case of fetal sick sinus syndrome (SSS) caused by pulmonary stenosis regurgitation (PSR) that spontaneously resolved during pregnancy. Case Report A 29-year-old woman was referred to our hospital at 21 weeks of gestation for persistent fetal bradycardia. Fetal echocardiography revealed PSR and ventricular septal defect (VSD). The ventricular rate was 60 to 70 beats/minute with 1:1 atrioventricular conduction. Thus, congenital SSS owing to PSR was suspected. During pregnancy, fetal SSS spontaneously resolved at 28 weeks of gestation despite persistent PSR. The ventricular rate was increased to approximately 120 beats/minute with regular rhythm. A 2,390-g male neonate was delivered via Caesarean section at 38 weeks of gestation. Consequently, detailed echocardiography revealed PSR and VSD without SSS. Conclusion Although fetal PSR can cause fetal SSS owing to immaturity at an earlier gestational age, SSS might be spontaneously resolved by fetal heart development as pregnancy progresses.

Amnion-derived cells as a reliable resource for next-generation regenerative medicine.

The placenta is composed of the amnion, chorionic plate, villous and smooth chorion, decidua basalis, and umbilical cord. The amnion is a readily obtainable source of a large number of cells and cell types, including epithelium, mesenchyme, and endothelium, and is thus an allogeneic resource for regenerative medicine. Endothelial cells are obtained from large arteries and veins in the amniotic membrane as well as the umbilical cord. The amnion-derived cells exhibit transdifferentiation capabilities, including chondrogenesis and cardiomyogenesis, by introduction of transcription factors, in addition to their original and potential phenotypes. The amnion is also a source for production of induced pluripotent stem cells (AM-iPSCs). The AM-iPSCs exhibit stable phenotypes, such as multipotency and immortality, and a unique gene expression pattern. Through the use of amnion-derived cells, as well as other placenta-derived cells, preclinical proof of concept has been achieved in a mouse model of muscular dystrophy.

Characterization of a Small Supernumerary Marker Chromosome Derived from Xq28 and 14q11.2 Detected Prenatally.

We present the characterization of a case with a small supernumerary marker chromosome (sSMC) detected prenatally derived from Xq28 and 14q11.2 maternal translocation. A 33-year-old Japanese woman, primigravida, underwent amniocentesis because of fetal growth restriction and fetal structural abnormality at 30 weeks of gestation. The fetal karyotype was identified as 47,XY,+mar. Additionally, the single nucleotide polymorphism array analysis revealed copy number gains at Xq28 and 14q11.2. A male infant, weighing 1,391 g, was delivered at term by cesarean section. Maternal and paternal karyotypes were 46,X,t(X; 14)(q28; q11) and 46,XY, respectively. These findings indicated that the sSMC might have originated from chromosome disjunction at a ratio of three to one. Here we describe a case with an sSMC derived from Xq28 and 14q11.2. Our findings suggest that this sSMC is most likely pathogenic. The collection of additional cases may be required.

Fetal Therapy Model of Myelomeningocele with Three-Dimensional Skin Using Amniotic Fluid Cell-Derived Induced Pluripotent Stem Cells.

Myelomeningocele (MMC) is a congenital disease without genetic abnormalities. Neurological symptoms are irreversibly impaired after birth, and no effective treatment has been reported to date. Only surgical repairs have been reported so far. In this study, we performed antenatal treatment of MMC with an artificial skin using induced pluripotent stem cells (iPSCs) generated from a patient with Down syndrome (AF-T21-iPSCs) and twin-twin transfusion syndrome (AF-TTTS-iPSCs) to a rat model. We manufactured three-dimensional skin with epidermis generated from keratinocytes derived from AF-T21-iPSCs and AF-TTTS-iPSCs and dermis of human fibroblasts and collagen type I. For generation of epidermis, we developed a protocol using Y-27632 and epidermal growth factor. The artificial skin was successfully covered over MMC defect sites during pregnancy, implying a possible antenatal surgical treatment with iPSC technology.

Prenatal Diagnosis of Isolated Agnathia-Otocephaly: A Case Report and Review of the Literature.

Agnathia is a rare disease characterized by the absence of a mandible. Few cases of prenatally diagnosed isolated agnathia have been reported. We present a case report and review of the literature of prenatally diagnosed agnathia. A 38-year-old woman (gravida 0, para 0) was referred to our hospital at 28 weeks and 3 days of gestation for fetal evaluation because of polyhydramnios and suspected facial anomalies. Three-dimensional ultrasonography and MRI indicated agnathia. Premature rupture of the membranes occurred before the parents could reach a decision on the postnatal treatment. We performed emergency cesarean section on the second day of the 33rd week of gestation. The neonate was deemed nonresuscitable and he died of airway obstruction shortly after birth. Because agnathia is associated with very poor prognosis, accurate prenatal diagnosis and detailed counseling should be promptly provided before unexpected delivery to the parents for the determination of postnatal treatment.