Dynamic acetylation profile during mammalian neurulation.

BACKGROUND: Neural tube defects (NTDs) result from failure of neural tube closure during embryogenesis. These severe birth defects of the central nervous system include anencephaly and spina bifida, and affect 0.5-2 per 1,000 pregnancies worldwide in humans. It has been demonstrated that acetylation plays a pivotal role during neural tube closure, as animal models for defective histone acetyltransferase proteins display NTDs. Acetylation represents an important component of the complex network of posttranslational regulatory interactions, suggesting a possible fundamental role during primary neurulation events. This study aimed to assess protein acetylation contribution to early patterning of the central nervous system both in human and murine specimens. METHODS: We used both human and mouse (Cited2 -/- ) samples to analyze the dynamic acetylation of proteins during embryo development through immunohistochemistry, western blot analysis and quantitative polymerase chain reaction. RESULTS: We report the dynamic profile of histone and protein acetylation status during neural tube closure. We also report a rescue effect in an animal model by chemical p53 inhibition. CONCLUSIONS: Our data suggest that the p53-acetylation equilibrium may play a role in primary neurulation in mammals.

Fetal pancreatic Langerhans islets size in pregnancies with metabolic disorders.

Objective: Metabolic disorders are a pandemic and increasing health problem. Women of childbearing age may also be affected, thus an abnormal metabolism may interfere with pregnancy short- and long-term outcomes, harming both mother and child. In the context of an abnormal maternal and intrauterine metabolic milieu the development of fetal organs, including pancreas, may be affected. Aim: To investigate the effects of pregnancy metabolic disorders on the morphology of pancreatic Langerhans islets in human late-third trimester stillborn fetuses. Methods: Samples from fetal pancreas underwent a quantitative histological evaluation to detect differences between pregnancy with (cases, n = 9) or without (controls, n = 6) abnormal metabolism. Results: Results show that the islets size increases in fetuses from dysmetabolic pregnancies and that this increment is related to both beta-cell hyperplasia and hypertrophy. Moreover, according to pregnancy and fetal metabolic disorders, a threshold of abnormal size of the islets has been identified. Above this threshold the size of fetal pancreatic Langerhans islets should be considered excessively increased. Conclusion: The study suggests that an accurate fetal pancreas analysis supplies an important tool in stillborn fetus, to discover metabolic disturbances that should be kept in mind and managed in future pregnancies.

Gestational diabetes affects fetal autophagy.

Autophagy is a catabolic process involved in the preservation of energy homeostasis and its dysregulation has been implicated in the development of metabolic disorders, including diabetes mellitus. Gestational diabetes mellitus represents a risk for fetal morbidity and mortality. The present study focuses on the autophagy process in human diabetic placenta and fetal pancreas, compared with controls. Analysis of the autophagy markers LC3, Beclin-1 and p62 suggests an impairment of the autophagy process in diabetic placentas. Results indicate an association between gestational diabetes and autophagy, emphasizing the importance of unravelling the mechanisms regulating this relationship.

Inflammation modulates LC3 expression in human preterm delivery.

OBJECTIVE: Autophagy is an inducible intracellular process acting under stressor conditions, such as infections, inflammation and hypoxia. The aim of the present study was to analyze autophagy expression in preterm delivered human placenta. METHODS: Autophagy marker LC3 was analyzed in 25 consecutive human placentas delivered before 34 weeks of gestation, analyzed by immunohistochemistry, immunofluorescence and quantitative real-time PCR, according to the histologic classification of preterm delivery (PTD) (cases with or without placental inflammatory lesions). RESULTS: LC3 expression was observed both in cases with and without inflammatory lesions. In cases with histological inflammation, strong immunoreactivity for LC3 autophagic marker was observed in the inflammatory cell infiltration composed by neutrophils. In all PTD cases, trophoblastic cells in chorion laeve express LC3, with variable staining intensity: a significant reduction of LC3 expression was observed in chorion laeve of PTD with histological inflammation compared to PTD without inflammatory lesions. Moreover, the decrement of LC3 staining was observed to be associated to the increasing severity of the histological signs of fetal inflammatory response. CONCLUSIONS: Our data show that the expression of LC3 varies depending on different histological features, indicating an interesting and possibly clinically relevant relation between autophagy expression levels and the inflammatory status.

Histopathology and molecular characterisation of intrauterine-diagnosed congenital craniopharyngioma.

PURPOSE: Adamantinomatous craniopharyngiomas (aCPs) are complex epithelial neoplasms that arise from the progenitors of the pituitary gland. Although benign, these tumours can be locally aggressive invading vital neighbouring structures such as the hypothalamus, the cranial and optic nerves. Congenital forms of aCPs diagnosed during foetal development are very rare. The purpose of this article is to present with a histopathological and molecular characterisation of congenital craniopharyngioma. METHODS: Here we report a case of in utero diagnosed aCP, detected at 21 weeks of gestation by ultrasound, visualised by MRI at 22 weeks and histologically diagnosed at 23 weeks. We provide with histopathological characterisation of rare form of congenital aCPs. RESULTS: Detailed examination of the tumour reveals the classical histological hallmarks of aCPs with the presence of stellate reticulum, palisading epithelium, wet keratin and calcification deposits. The tumour demonstrated complete absence of all pituitary hormones and the absence of the neuroendocrine marker, synaptophysin. Immunohistochemistry against ß-catenin revealed occasional cells with nuclear-ß-catenin localisation and the presence of pituitary progenitors positive for SOX9 and SOX2. Targeted Sanger sequencing revealed no genetic variants in oncogenes CTNNB1 and BRAF, previously associated with CP. CONCLUSIONS: In this article, we provide with in-depth molecular and histological characterisation of in utero aCP due to an unknown driving mutation that could represent a sub-cohort of congenital aCPs.

Cell death and cell proliferation in human spina bifida.

BACKGROUND: Spina bifida is a multifactorial congenital malformation of the central nervous system. The aim of this study was to ascertain the relevance of cell death/proliferation balance in human spina bifida and to assess autophagy distribution and levels during embryo-fetal development in neural tissue. METHODS: Five human cases with myelomeningocoele were compared with 10 healthy human controls and LC3 protein expression was also analyzed in mouse embryos. Cell death was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) assay; cell proliferation was studied by counting Ki67-positive cells, and autophagy was assessed by observing the presence of LC3 punctuate dots. RESULTS: Comparing human cases and controls (13 to 21 weeks of gestation), we observed a significant increase in TUNEL-positive cells in human spina bifida associated with a significantly decreased proliferation rate, indicating an alteration of the physiological cell rate homeostasis. LC3 distribution was found to be spatiotemporally regulated in both human and murine embryo-fetuses: in early pregnancy a diffuse and ubiquitous LC3 signal was detected. After neural tube closure, an intense LC3-positive signal, normally associated to extra energy requirement, was confined to the Lissauer's tract, the dorsolateral spinal zone containing centrally projecting axons from dorsal root ganglia, at any medullar levels. LC3 signal disappeared from 12 weeks of gestation. CONCLUSION: In conclusion, this study confirms the fundamental role of cell death/proliferation balance during central nervous system development and reports the changing expression of LC3 protein in mouse and human neural tube. Birth Defects Research (Part A) 106:104-113, 2016. © 2015 Wiley Periodicals, Inc.

Autophagy in Normal and Abnormal Early Human Pregnancies.

Autophagy is an inducible catabolic process by which cells degrade and recycle materials to survive stress, starvation, and hypoxia. The aim of this study was to evaluate autophagy at the fetal-maternal interface, to assess autophagy involvement during the early phase of human gestation, and to explore autophagic modification in case of early abnormal pregnancy outcome. Specimens were collected from first-trimester normal gestations undergoing legal termination of pregnancy and first-trimester sporadic spontaneous miscarriages. Autophagy was studied in villous and decidual samples by transmission electron microscopy, immunohistochemistry, immunofluorescence, and Western blotting. Autophagy markers were found in cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, and decidual stromal cells. Autophagy is physiologically involved in early normal gestation. Compared with normal pregnancy, spontaneous miscarriage presents an increase in autophagy expression in villous specimens due to an increment in concentration of autophagic vacuole in syncytiotrophoblast, suggesting a cytoprotective mechanism of the cells to respond to microenvironmental challenge.

Autophagy and human parturition: evaluation of LC3 expression in placenta from spontaneous or medically induced onset of labor.

Induction of labor is one of the most used procedures in obstetrics, performed to achieve vaginal delivery through cervical ripening and stimulation of uterine contractions. We investigated the impact of induction of labor upon placental autophagy, a catabolic pathway activated in response to alteration of the physiological intracellular conditions. We collected 28 singleton placentas at the time of uncomplicated term vaginal delivery (7 spontaneous onset of labor, 21 induced labor). Autophagy was evaluated by immunohistochemistry, immunofluorescence, and immunoblotting. No significant difference in the autophagy expression was found between spontaneous or induced onset of labor. We found an inverse relationship between autophagy expression and the maternal prepregnancy body mass index, irrespective of the mode of labor onset. This result could be related to the nutritional maternal habits before and throughout pregnancy rather than rapid metabolic changes during labor.

SNAT2 expression and regulation in human growth-restricted placentas.

BACKGROUND: Amino acid placental delivery is reduced in human intrauterine growth-restricted (IUGR) fetuses, and the activity of placental amino transporters has been consistently shown to be decreased in in vitro studies. We hypothesized lower placental expression and localization of sodium-coupled neutral amino acid transporter 2 (SNAT2 (also known as SLC38A2)), altered levels of intron-1 methylation, and altered distribution of single-nucleotide polymorphisms in human IUGR vs. normal pregnancies. METHODS: We studied 88 IUGR and 84 control placentas from singleton pregnancies at elective caesarean section. SNAT2 expression was investigated by real-time PCR and immunohistochemistry. Intron-1 methylation levels were analyzed by pyrosequencing, and single-nucleotide polymorphism distribution was analyzed by allelic discrimination. RESULTS: mRNA levels were significantly decreased in IUGR placentas with reduced umbilical blood flows. Syncytiotrophoblast immunostaining was lower in IUGR placentas than in control placentas. Methylation levels were steadily low in both IUGR and control placentas. SNP genotype and allele frequencies did not differ between the two groups. CONCLUSION: This is the first study investigating SNAT2 expression and regulation mechanisms in human IUGR placentas. We confirm previous results obtained in rats and cell cultures that support the fundamental role of SNAT2 in fetal growth and well-being, as well as a possible role of oxygen levels in regulating SNAT2 expression, indicating the relevance of hypoxia in IUGR.

An imbalance of COX level is not related to placental abruption.

AIMS: Muscularised basal plate arteries (MA) or chorioamnionitis (CA) are often present in placental abruption. The aim of this study was to evaluate the placental expression of COX 1 and COX 2 in cases of placental abruption with MA or CA hypothesising that an imbalance in COX placental expression might be implicated in its pathogenesis. METHODS: COX 1 and COX 2 placental immunostaining was analysed in 16 placentas with abruption (nine with MA, seven with CA), in 26 normal placentas and in 10 gestational age-matched MA or CA cases without abruption. RESULTS: COX 1 and COX 2 protein expression was observed in all cases, both in placental abruption and in normal placentas. No differences in distribution of immunoreactivity were observed either between cases and controls or between MA and CA. The mean COX 1 ratio between COX-positive cells and all stromal cells was significantly lower in placental abruption with MA (0.14±0.05) when compared with cases with CA (0.35±0.06) and normal placenta (0.23±0.02; p<0.001). The mean COX 2 ratio was lower in placental abruption with MA than in normal placenta (0.09±0.06 vs 0.18±0.05: p<0.001). In contrast, no difference in COX 1 and COX 2 ratio was observed between MA cases with or without abruption and between CA cases with or without abruption. CONCLUSIONS: It is hypothesised that an imbalance of normal COX level may be present in cases with MA and CA but it is not related to placental abruption.