Quantitative Proteomics of Maternal Blood Plasma in Isolated Intrauterine Growth Restriction.

Intrauterine growth restriction (IUGR) remains a significant concern in modern obstetrics, linked to high neonatal health problems and even death, as well as childhood disability, affecting adult quality of life. The role of maternal and fetus adaptation during adverse pregnancy is still not completely understood. This study aimed to investigate the disturbance in biological processes associated with isolated IUGR via blood plasma proteomics. The levels of 125 maternal plasma proteins were quantified by liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) with corresponding stable isotope-labeled peptide standards (SIS). Thirteen potential markers of IUGR (Gelsolin, Alpha-2-macroglobulin, Apolipoprotein A-IV, Apolipoprotein B-100, Apolipoprotein(a), Adiponectin, Complement C5, Apolipoprotein D, Alpha-1B-glycoprotein, Serum albumin, Fibronectin, Glutathione peroxidase 3, Lipopolysaccharide-binding protein) were found to be inter-connected in a protein-protein network. These proteins are involved in plasma lipoprotein assembly, remodeling, and clearance; lipid metabolism, especially cholesterol and phospholipids; hemostasis, including platelet degranulation; and immune system regulation. Additionally, 18 proteins were specific to a particular type of IUGR (early or late). Distinct patterns in the coagulation and fibrinolysis systems were observed between isolated early- and late-onset IUGR. Our findings highlight the complex interplay of immune and coagulation factors in IUGR and the differences between early- and late-onset IUGR and other placenta-related conditions like PE. Understanding these mechanisms is crucial for developing targeted interventions and improving outcomes for pregnancies affected by IUGR.

A Case of Pregnancy Complicated by Primary Hyperparathyroidism Due to a Parathyroid Adenoma.

BACKGROUND Primary hyperparathyroidism is most common in women during the menopause and its occurrence in pregnant women is rare. However, because neonatal mortality is associated with maternal hyperparathyroidism, early diagnosis is essential. This report describes the case of a late diagnosis of primary hyperparathyroidism in a 28-year-old pregnant woman and describes the effects on the mother and neonate. CASE REPORT During her second pregnancy, a 28-year-old woman presented with symptoms of general weakness, bone and joint pain, multiple fractures with bone deformity, muscle weakness, and gait disturbance. Due to the high risk of perinatal pathology, a cesarean section was performed. Several weeks later, she underwent thoracoscopic removal of an ectopic parathyroid gland located at the aortic arch. Hypocalcemia in the newborn infant required treatment with calcium and magnesium supplements. CONCLUSIONS This case demonstrates that primary hyperparathyroidism during pregnancy requires timely diagnosis and treatment to reduce potential maternal and fetal complications. Screening for primary hyperparathyroidism should be undertaken in pregnant women with any symptoms associated with hypercalcemia. Treatment should be individualized and includes conservative management, parathyroidectomy in the second trimester, or parathyroidectomy performed in the early postpartum period.

Synthetic osmotic dilators for cervical preparation prior to abortion-An international multicentre observational study.

OBJECTIVES: To evaluate the outcomes with the use of Dilapan-S for cervical preparation prior to medical or surgical abortion. STUDY DESIGN: International, multicentre, prospective observational study in women between 6 + 0-24 + 0 weeks' gestation. The study was conducted across 7 study sites in 4 countries, between 1/5/2015 to 31/12/2016. The primary outcomes studied were the number of dilators used and the duration required for cervical preparation prior to abortion. Secondary outcomes were complications of dilator use and infection. Participants were followed-up for 4 weeks post procedure to capture any delayed complications. RESULTS: A total of 483 women were enrolled with 439 women eligible for analysis. Medical abortion was performed in 38% (n = 165) women and surgical abortion in 62% (n = 274). For medical abortions and surgical abortions, an average of 3 osmotic dilators for time interval of 4-7 hours provided effective cervical preparation. Medical abortions were performed as day-case procedures (<12 h) in 81% of women. There was no difference in using either adjunctive misoprostol or Dilapan-S followed by misoprostol for cervical ripening effect to achieve complete medical abortion. Dilapan-S permitted surgical abortions to be performed as same-day procedures (<12 h), in 85% of women regardless of gestational age and without the need to use adjunctive or additional misoprostol. There were no serious adverse events reported with the use of Dilapan-S, including in women with a previous caesarean section. The overall infectious morbidity was 0.9% of cases with no causal relationship with the use of synthetic osmotic dilator use (for a length <24 h). In addition, Dilapan-S was reported as easy to insert and remove in over 90% of women. CONCLUSION: Dilapan-S is a safe and effective method for cervical preparation for medical and surgical abortions up to 24 weeks' gestation. It allows medical and surgical abortions to be performed as day case procedures and is associated with a low complication rate. Future research should aim at directly comparing Dilapan-S and preferred pharmacological agents in a randomised controlled trial.

Ultrastructural and Immunohistochemical Features of Telocytes in Placental Villi in Preeclampsia.

A new cell type, interstitial Cajal-like cell (ICLC), was recently described in different organs. The name was recently changed to telocytes (TCs), and their typical thin, long processes have been named telopodes (Tp). TCs regulate the contractile activity of smooth muscle cells and play a role in regulating vessel contractions. Although the placenta is not an innervated organ, we believe that TCs are present in the placenta. We studied placenta samples from physiological pregnancies and in different variants of preeclampsia (PE). We examined these samples using light microscopy of semi-thin sections, transmission electron microscopy, and immunohistochemistry. Immunohistochemical examination was performed with primary antibodies to CD34, CD117, SMA, and vimentin, and TMEM16a (DOG-1), the latter was used for the diagnosis of gastrointestinal stromal tumours (GIST) consisting of TCs. We have identified a heterogenetic population of ТСs in term placentas, as these cell types differed in their localization, immunophenotype and ultrastructural characteristics. We assume TMEM16a could be used as the marker for identification of TCs. In PE we have revealed telocyte-like cells with ultrastructural signs of fibrocytes (significant process thickening and the granular endoplasmic reticulum content was increased) and a loss of TMEM16a immunohistochemical staining.

The role of extracellular inducer of matrix metalloproteinases in premature rupture of membranes.

OBJECTIVE: To investigate the role of matrix metalloproteinases (MMP-2, MMP-9) and their inducer (CD147) in premature rupture of membranes (PROM) at term labor. METHODS: In a cross-sectional study, 24 women aged 19-39, with 37-40-week pregnancy, and no clinical and histological signs of chorioamnionitis, were divided into two groups with and without PROM. The histological and immunohistochemical study of the fetal membranes was performed with polyclonal rabbit antibodies to MMP-2/MMP-9 and monoclonal rabbit antibodies to CD147. RESULTS: The analysis of MMP revealed the increase of MMP-9 expression in the amniotic epithelium during premature membrane rupture both in rupture area, and beyond it, and increased MMR-2 expression in the mesodermal cells. We also found high level of CD147 in the amniotic epithelium in PROM group. The above-mentioned changes were found in all areas of fetal membranes, regardless of the rupture localization. CONCLUSIONS: The study results demonstrate the increased expression of MMR-2 and MMR-9, which regulate the catabolism of fetal membrane extracellular matrix proteins, in amniotic membranes of women with PROM at term labor. The increased expression of CD147 may be one of the mechanisms triggering PROM in the absence of infection.

Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.

Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in vitro modelling of human genetic disorders for pathogenic investigations and therapeutic screens. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging owing to the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease characterized by pathological fatty infiltration and cardiomyocyte loss predominantly in the right ventricle, which is associated with life-threatening ventricular arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly in PKP2, encoding plakophilin-2 (ref. 9). The median age at presentation of ARVD/C is 26 years. We used previously published methods to generate iPSC lines from fibroblasts of two patients with ARVD/C and PKP2 mutations. Mutant PKP2 iPSC-CMs demonstrate abnormal plakoglobin nuclear translocation and decreased ß-catenin activity in cardiogenic conditions; yet, these abnormal features are insufficient to reproduce the pathological phenotypes of ARVD/C in standard cardiogenic conditions. Here we show that induction of adult-like metabolic energetics from an embryonic/glycolytic state and abnormal peroxisome proliferator-activated receptor gamma (PPAR-γ) activation underlie the pathogenesis of ARVD/C. By co-activating normal PPAR-alpha-dependent metabolism and abnormal PPAR-γ pathway in beating embryoid bodies (EBs) with defined media, we established an efficient ARVD/C in vitro model within 2 months. This model manifests exaggerated lipogenesis and apoptosis in mutant PKP2 iPSC-CMs. iPSC-CMs with a homozygous PKP2 mutation also had calcium-handling deficits. Our study is the first to demonstrate that induction of adult-like metabolism has a critical role in establishing an adult-onset disease model using patient-specific iPSCs. Using this model, we revealed crucial pathogenic insights that metabolic derangement in adult-like metabolic milieu underlies ARVD/C pathologies, enabling us to propose novel disease-modifying therapeutic strategies.

Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells.

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of selection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line-specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming-specific epigenetic signature comprised of nine aberrantly methylated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state.

Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

DMSO-Free Programmed Cryopreservation of Fully Dissociated and Adherent Human Induced Pluripotent Stem Cells.

THREE MODES FOR CRYOPRESERVATION (CP) OF HUMAN IPSC CELLS HAVE BEEN COMPARED: STD: standard CP of small clumps with 10% of CPA in cryovials, ACC: dissociation of the cells with Accutase and freezing in cryovials, and PLT: programmed freezing of adherent cells in plastic multiwell dishes in a programmable freezer using one- and multistep cooling protocols. Four CPAs were tesetd: dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY). The cells in ACC and PLT were frozen and recovered after thawing in the presence of a ROCK inhibitor Y-27632 (RI). EG was less toxic w/o CP cryopreservation than DMSO and allowed much better maintenance of pluripotency after CP than PG or GLY. The cells were cryopreserved very efficiently as adherent cultures (+RI) in plates (5-6-fold higher than STD) using EG and a 6-step freezing protocol. Recovery under these conditions is comparable or even higher than ACC+RI. Conclusions. Maintenance of cell-substratum adherence is a favorable environment that mitigates freezing and thawing stresses (ComfortFreeze(®) concept developed by CELLTRONIX). CP of cells directly in plates in ready-to-go after thawing format for HT/HC screening can be beneficial in many SC-related scientific and commercial applications such as drug discovery and toxicity tests.

Compensatory growth mechanisms regulated by BMP and FGF signaling mediate liver regeneration in zebrafish after partial hepatectomy.

Here, we describe the zebrafish (Danio rerio) as a vertebrate model system to study liver regeneration with the added benefit of its powerful genetics and screening possibilities to uncover the molecular pathways underlying liver regeneration. We developed a partial hepatectomy (PH) protocol in zebrafish and investigated in detail the cellular and morphological changes during the process of liver regeneration. We show that the type of regenerative response is dependent on the size of the injury sustained by the zebrafish liver. Furthermore, we demonstrate for the first time that the mechanisms of liver regeneration in zebrafish after PH are strikingly similar to those of rodents and humans, with 100% recovery of the liver mass after 6-7 d postsurgery. This occurs via compensatory growth mediated by proliferation of hepatocytes throughout the entire liver remnant. By analyzing transgenic fish expressing dominant-negative forms of either bone morphogenetic protein (BMP) receptor or fibroblast growth factor (FGF) receptor 1, we demonstrate that the BMP and FGF signaling pathways are crucial regulators of the early events during liver regeneration after PH. Our study demonstrates that the mechanisms of liver regeneration in zebrafish are highly similar to the processes ongoing during mammalian liver regeneration and make the adult zebrafish a suitable model system to study the mechanisms of liver regeneration.

Gene replacement reveals a specific role for E-cadherin in the formation of a functional trophectoderm.

During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. Here, we analyzed to what extent E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, we introduced N-cadherin cDNA into the E-cadherin genomic locus. We show that the expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, we demonstrate that N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. Our results point to a specific and unique function for E-cadherin during mouse preimplantation development.

Isolation and characterization of highly radioresistant malignant hamster fibroblasts that survive acute gamma irradiation with 20 Gy.

To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK-) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK- cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D0 for PIC-20 cells was 7.4 +/- 0.2 Gy, which is three times higher than that for parental cells (2.5 +/- 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK- cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK- cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.

Expression of mammalian Rab Escort protein-1 and -2 in yeast Saccharomyces cerevisiae.

Rab GTPases are post-translationally geranylgeranylated on their C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase) and this modification is essential for their biological activity. Rab Escort Protein (REP) is a molecular chaperone that assists in the prenylation reaction carried out by RabGGTase. Mutations in the REP-1 gene lead to progressive retinal degradation and blindness in humans. Despite the significant interest in REP proteins, their preparative production remains challenging. We report here an inducible expression system for the production of REP-1 and REP-2 in yeast Saccharomyces cerevisiae at levels 3.5 and 2mg/L, respectively. The REP-1 was found to be toxic for yeast cells and its toxicity is proposed to be associated with the formation of unproductive Ypt:REP-1 complexes. To minimize the toxic effect of REP-1, the recombinant protein expression was induced at the end of the exponential phase. Under these conditions, the GAL1 promoter is no longer repressed due to exhaustion of glucose and utilization of ethanol as a carbon source. This expression procedure was successfully scaled up to 30L for both proteins. The REP-1 and REP-2 were purified using a combination of affinity and anion-exchange chromatography. Purified proteins were functionally active, as determined by a fluorescent Rab binding assay and in vitro prenylation. The reported procedure provides a reliable source of REP proteins for biochemical and structural studies.