The Association between Low Fetal Fraction of Non-Invasive Prenatal Testing and Adverse Pregnancy Outcomes for Placental Compromise.

(1) Background: Non-invasive prenatal testing (NIPT) is a screening test for fetal aneuploidy using cell-free fetal DNA. The fetal fragments (FF) of cell-free DNA (cfDNA) are derived from apoptotic trophoblast of the placenta. The level of fetal cfDNA is known to be influenced by gestational age, multiple pregnancies, maternal weight, and height. (2) Methods: This study is a single-center retrospective observational study which examines the relationship between the fetal fraction (FF) of cell-free DNA in non-invasive prenatal testing (NIPT) and adverse pregnancy outcomes in singleton pregnancies. A total of 1393 samples were collected between 10 weeks and 6 days, and 25 weeks and 3 days of gestation. (3) Results: Hypertensive disease of pregnancy (HDP) occurred more frequently in the low FF group than the normal FF group (5.17% vs. 1.91%, p = 0.001). Although the rates of small for gestational age (SGA) and placental abruption did not significantly differ between groups, the composite outcome was significantly higher in the low FF group (7.76% vs. 3.64%, p = 0.002). Furthermore, women who later experienced complications such as HDP or gestational diabetes mellitus (GDM) had significantly lower plasma FF levels compared to those without complications (p < 0.001). After adjustments, the low FF group exhibited a significantly higher likelihood of placental compromise (adjusted odds ratio: 1.946). (4) Conclusions: Low FF in NIPT during the first and early second trimesters is associated with adverse pregnancy outcomes, particularly HDP, suggesting its potential as a predictive marker for such outcomes.

Maternal Anemia during the First Trimester and Its Association with Psychological Health.

Anemia during pregnancy is known to be associated with an increased risk of antenatal and/or postnatal depression, as well as adverse pregnancy outcomes. However, there are few studies evaluating psychological health throughout the antepartum and postpartum periods in women with anemia in early pregnancy. This study analyzed data collected by the Korean Pregnancy Outcome Study, a multicenter prospective cohort study conducted in South Korea, to determine the impact of anemia during the first trimester on birth outcomes and maternal mental health during pregnancy and postpartum. Hemoglobin levels were measured during the first trimester, and psychological health was evaluated at 12, 24, and 36 gestational weeks and 4−6 weeks postpartum. Anxiety and depression were defined using the Hospital Anxiety and Depression Scale and the Edinburgh Postnatal Depression Scale, respectively. Among 4067 Korean participants, 119 (2.9%) were diagnosed with anemia during the first trimester. Incidences of anxiety and depression did not differ over the pregnancy period between those with and without anemia during the first trimester. However, postpartum anxiety and depression were significantly more common in participants with anemia than in those without (p < 0.05, both). Hence, obstetricians should pay attention to postpartum mental health in women with anemia during the first trimester.

Clinically significant maternal X chromosomal copy number variation detected by noninvasive prenatal test.

Maternal copy number variation (CNV), especially at the X chromosome is an important cause of false positive noninvasive prenatal test (NIPT) results for sex chromosomal aneuploidy. In addition, some maternal CNV can cause significant anomalies if the male fetus was inherited the X chromosome with CNV. During 1000 high risk Korean NIPT, we incidentally detected two cases of maternal X chromosomal CNV which can cause abnormal phenotype in a male fetus. The first false-positive NIPT case (47, XXY) was due to a maternal 0.5 Mb duplication at Xq28, including the MECP2 gene. The second is a case of an 8-Mb deletion on maternal Xq24q25, including GRIA3 and XIAP genes.

Evaluating the influence of climate change on the fate and transport of fecal coliform bacteria using the modified SWAT model.

Fecal coliform bacteria (FCB) contamination of natural waters is a serious public health issue. Therefore, understanding and anticipating the fate and transport of FCB are important for reducing the risk of contracting diseases. The objective of this study was to analyze the impacts of climate change on the fate and transport of FCB. We modified both the soil and the in-stream bacteria modules in the soil and water assessment tool (SWAT) model and verified the prediction accuracy of seasonal variability of FCB loads using observations. Forty bias-correcting GCM-RCM projections were applied in the modified SWAT model to examine various future climate conditions at the end of this century (2076-2100). Lastly, we also compared the variability of FCB loads under current and future weather conditions using multi-model ensemble simulations (MMES). The modified SWAT model yielded a satisfactory performance with regard to the seasonal variability of FCB amounts in the soil and FCB loading to water bodies. The modified SWAT model presented substantial proliferation of FCB in the soil (30.1%-147.5%) due to an increase in temperature (25.1%). Also, increase in precipitation (53.3%) led to an increase in FCB loads (96.0%-115.5%) from the soil to water body. In the in-stream environment, resuspension from the stream bed was the dominant process affecting the amount of FCB in stream. Therefore, the final FCB loads increased by 71.2% because of the growing peak channel velocity and volume of water used due to an increase in precipitation. Based on the results of MMES, we concluded that the level of FCB would increase simultaneously in the soil as well as in stream by the end of this century. This study will aid in understanding the future variability of FCB loads as well as in preparing an effective management plan for FCB levels in natural waters.

Functional analysis of cell-free RNA using mid-trimester amniotic fluid supernatant in pregnancy with the fetal growth restriction.

The prediction and monitoring of fetal growth restriction (FGR) fetuses has become with the use of ultrasound. However, these tools lack the fundamental evidence for the growth of fetus with FGR excluding pathogenic factors.Amniotic fluid samples were obtained from pregnant women for fetal karyotyping and genetic diagnosis at 16 to 19 weeks of gestation. For this study, 15 FGR and 9 control samples were selected, and cell-free fetal RNA was isolated from each supernatant of the amniotic fluid for microarray analysis.In this study, 411 genes were differentially expressed between the FGR and control group. Of these genes, 316 genes were up-regulated, while 95 genes were down-regulated. In terms of gene ontology, the up-regulated genes were highly related to metabolic process as well as protein synthesis, while the down-regulated genes were related to receptor activity and biological adhesion. In terms of tissue-specific expression, the up-regulated genes were involved in various organs while down-regulated genes were involved only in the brain. In terms of organ-specific expression, many genes were enriched for B-cell lymphoma, pancreas, eye, placenta, epithelium, skin, and muscle. In the functional significance of gene, low-density lipoprotein receptor-related protein 10 (LRP10) was significantly increased (6-fold) and insulin-like growth factor (IGF-2) was dramatically increased (17-fold) in the FGR cases.The results show that the important brain-related genes are predominantly down-regulated in the intrauterine growth restriction fetuses during the second trimester of pregnancy. This study also suggested possible genes related to fetal development such as B-cell lymphoma, LRP10, and IGF-2. To monitor the fetal development, further study may be needed to elucidate the role of the genes identified.

Hypoxia-induced downregulation of XIAP in trophoblasts mediates apoptosis via interaction with IMUP-2: implications for placental development during pre-eclampsia.

The regulation of trophoblast apoptosis is essential for normal placentation, and increased placental trophoblast cell apoptosis is the cause of pathologies such as intrauterine growth retardation (IUGR) and pre-eclampsia. X-linked inhibitor of apoptosis (XIAP) is expressed in trophoblasts, but little is known about the role of XIAP in placental development. In the present study, the function of XIAP in the placenta and in HTR-8/SVneo trophoblasts under hypoxic conditions was examined. In addition, the correlation between XIAP and immortalization-upregulated protein-2 (IMUP-2) was demonstrated in HTR-8/SVneo trophoblasts under hypoxia, based on a previous study showing that increased IMUP-2 induces trophoblast apoptosis and pre-eclampsia. XIAP was downregulated in pre-eclamptic placentas (P < 0.05). In HTR-8/SVneo trophoblasts, XIAP expression was decreased and the expression of apoptosis-related genes was increased in response to hypoxia. Ectopic expression of hypoxia inducible factor (HIF)-1α in HRT-8 SV/neo cells induced the nuclear translocation of XIAP and alterations of XIAP protein stability. Furthermore, hypoxia induced nuclear translocated XIAP co-localized with upregulated IMUP-2 in trophoblast nuclei, and the interaction between XIAP and IMUP-2 induced apoptosis in HRT-8 SV/neo cells. The present results suggest that hypoxia-induced down-regulation of XIAP mediates apoptosis in trophoblasts through interaction with increased IMUP-2, and that this mechanism underlies the pathogenesis of pre-eclampsia.

Prognostic factors for treatment outcomes in transient tachypnea of the newborn.

BACKGROUND: Transient tachypnea of the newborn (TTN) is a clinical syndrome associated with respiratory distress usually seen shortly after delivery in infants. This study aims to determine the risk factors predicting treatment outcomes in infants with TTN. METHODS: Data from 236 infants diagnosed with TTN during the study period were evaluated retrospectively. Logistic regression analyses were performed to select significant risk factor for prognosis (prolonged oxygen therapy, application of mechanical ventilator, and prolonged hospital stay) of TTN among components of clinical variables. RESULTS: Of the 236 TTN infants, 111 (47.0%) infants were delivered via cesarean section (CS) without labor, 29 (12.3%) infants were delivered via CS with labor, and 96 (40.7%) were delivered via vaginal birth. Lower Apgar score at 1 min (OR: 3.03; 95%CI: 1.25-7.36) and lower umbilical artery pH (OR: 4.00; 95%CI 1.55-10.49) were associated with a significantly increased risk for mechanical ventilator care. Also, late-preterm delivery (OR: 4.70; 95%CI: 2.11-10.49) was independently associated with risk of prolonged duration of hospital stay. CONCLUSIONS: Late-preterm delivery, lower initial umbilical artery pH (<7.25), and lower Apgar score at 1 min were independently associated with poor prognostic treatment outcomes in infants with TTN.

Human chorionic-plate-derived mesenchymal stem cells and Wharton's jelly-derived mesenchymal stem cells: a comparative analysis of their potential as placenta-derived stem cells.

Placenta-derived stem cells (PDSCs) have gained interest as an alternative source of stem cells for regenerative medicine because of their potential for self-renewal and differentiation and their immunomodulatory properties. Although many studies have characterized various PDSCs biologically, the properties of the self-renewal and differentiation potential among PDSCs have not yet been directly compared. We consider the characterization of chorionic-plate-derived mesenchymal stem cells (CP-MSCs) and Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) among various PDSCs and the assessment of their differentiation potential to be important for future studies into the applicability and effectiveness of PDSCs in cell therapy. In the present study, the capacities for self-renewal and multipotent differentiation of CP-MSCs and WJ-MSC isolated from normal term placentas were compared. CP-MSCs and WJ-MSCs expressed mRNAs for the pluripotent stem cell markers Oct-4, Nanog, and Sox-2. Additionally, HLA-G for immunomodulatory effects was found to be expressed at both the mRNA and protein levels in both cell types. The CP-MSCs and WJ-MSCs also had the capacities to differentiate into cells of mesodermal (adipogenic and osteogenic) and endodermal (hepatogenic) lineages. Expression of adipogenesis-related genes was higher in CP-MSCs than in WJ-MSCs, whereas WJ-MSCs accumulated more mineralized matrix than CP-MSCs. The WJ-MSCs expressed more of CYP3A4 mRNA, a marker for mature hepatocytes, than CP-MSCs. Thus, we propose that CP-MSCs and WJ-MSCs are useful sources of cells for appropriate clinical applications in the treatment of various degenerative diseases.

ITI-H4, as a biomarker in the serum of recurrent pregnancy loss (RPL) patients.

Recurrent pregnancy loss (RPL) is defined as at least three pregnancy losses in series prior to the 20-28 weeks of pregnancy. There are several etiological factors associated with immunology, anatomy, endocrinology, genetic, infection, chromosomal abnormalities, and environmental factors contributing to the condition. The aim of this study was to identify RPL associated factors in human blood using proteomics. Since it is difficult to obtain tissues or follicular fluids, we used blood samples from normal and RPL patients to conduct a comparative proteomic study. Three RPL blood samples and one cocktailed blood sample from 3 normal women were used. We performed 2-DE and selected spots were analyzed with MALDI-TOF/MS. In the three RPL blood samples, 2-DE analysis revealed 549, 563 and 533 spots to be differentially expressed, respectively. Through a comparative analysis between the control and RPL, 21 spots were shown to be differentially expressed. Of these, 5 proteins were confirmed by Western blot analysis. One of these proteins, ITI-H4 (inter-α trypsin inhibitor-heavy chain 4), was weakly expressed at a molecular weight of 120 kDa, but was highly expressed at a modified molecular weight of 36 kDa in RPL patients. These findings suggest that ITI-H4 expression may be used as a biomarker, which could facilitate the development of novel diagnostic and therapeutic tools.

Comparative proteomic analysis of human amniotic fluid supernatants with Down syndrome using mass spectrometry.

Down syndrome (DS) is an abnormality of the 21st chromosome that commonly occurs in children born to advanced age women. Amniotic fluid (AF) is usually collected from such women for prenatal diagnosis. This study analyzed human AF supernatants (AFS) by mass spectrometric (MS) approach to search for candidate biomarkers of DS pregnancy. AFS were collected from advanced age pregnant women at 16-18th weeks of gestation by amniocentesis for cytogenetic analysis. AFS from pregnancies carrying DS (n=4) or chromosomally normal (n=6) fetuses, as revealed by cytogenetic analysis, were then subjected to global protein profiling by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Affinity chromatography was applied prior to LC-ESI-MS/MS to minimize the masking effect of highly abundant albumin and immunoglobulin and thereby, increased the diversity of identified proteins. Hereby, at least 30 AFS proteins were newly identified and 44 AFS proteins were found to be differentially expressed between DS and normal cases. Six of these proteins were unique to DS cases while 11 proteins were unique to chromosomally normal cases. In addition, 19 AFS proteins were down-regulated and 8 were up-regulated in DS cases with varying fold changes. A western blot analysis confirmed the LC-ESI-MS/MS data that combined detection of Apolipoprotein A-II (apo A-II) and alpha-fetoprotein (AFP) could be a potential tool for diagnosing DS cases.

Increased immortalization-upregulated protein 2 (IMUP-2) by hypoxia induces apoptosis of the trophoblast and pre-eclampsia.

In regulation of the developmental process, the balance between cellular proliferation and cell death is critical. Placental development tightly controls this mechanism, and increased apoptosis of placental trophoblasts can cause a variety of gynecological diseases. Members of the immortalization-upregulated protein (IMUP) family are nuclear proteins implicated in SV40-mediated immortalization and cellular proliferation; however, the mechanisms by which their expression is regulated in placental development are still unknown. We compared IMUP-2 expression in normal and pre-eclamptic placental tissues and evaluated the function of IMUP-2 in HTR-8/SVneo trophoblast cells under hypoxic conditions. IMUP-2 was expressed in syncytiotrophoblasts and syncytial knots of the placental villi. IMUP-2 expression was significantly higher in preterm pre-eclampsia patients than in patients who went to term (P < 0.001); however, we observed no differences in IMUP-2 expression between normal term patients with and without pre-eclampsia. Hypoxic conditions increased apoptosis of HTR8/SVneo trophoblast cells and induced IMUP-2 expression. Also, apoptosis of HTR-8/SVneo trophoblast cells was increased after IMUP-2 gene transfection. These results suggest that IMUP-2 expression is specifically elevated in preterm pre-eclampsia and under hypoxic conditions, and that IMUP-2 induces apoptosis of the trophoblast. Therefore, IMUP-2 might have functional involvement in placental development and gynecological diseases such as pre-eclampsia.

14-3-3 gamma is stimulated by IL-3 and promotes cell proliferation.

IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomics-based approaches, we identified 11 proteins. Of these, expression of 14-3-3gamma was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3gamma overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3gamma in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the up-regulation of Bax and Bad was seen with the ablation of 14-3-3gamma, resulting in cell death. These results indicate that deregulated expression of 14-3-3gamma may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.

Association study for single nucleotide polymorphisms in the CYP17A1 gene and polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) are characterized by excess androgen secretion and anovulatory infertility as a cause of follicular maturation arrest, and they are also associated with insulin resistance and obesity. Recently, it was suggested that one of the etiologies for PCOS is an abnormality of steroid hormones, and excessive secretion of androgen. The endoplasmic reticular cytochrome P450, 17alpha-hydroxylase (CYP17A), plays a key role in the mechanism of steroid hormones such as adrenal and gonadal steroid biosynthesis. Therefore, we studied the association between single nucleotide polymorphisms (SNPs) of the A1 allelic variant of the CYP17 gene and PCOS in a Korean population. The study recruited 134 Korean women with PCOS and 100 healthy women as controls. Using the HapAnalyzer, the genotype of the CYP17A1 polymorphism in PCOS and control patients were analyzed. We considered a p-value lower than 0.05 to be statistically significant. After genotypic analysis, we found seven SNPs of the CYP17A1 gene in a large population of subjects. The frequency of seven SNPs had no significant association with PCOS. However, one haplotype (ht3) had a p-value of p=0.001, suggesting that it may be associated with the pathogenesis of PCOS in a Korean population.

Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction.

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis.

ROC analysis of an erythroblast morphologic scoring system to improve identification of fetal cells in maternal blood.

OBJECTIVES: Nucleated red blood cells are used in research settings as a target cell type for investigations of noninvasive prenatal diagnosis, and these cells have a characteristic nuclear morphology and hemoglobin staining pattern that makes them distinguishable from maternal cells. Recently, we developed an erythroblast scoring system based on these characteristics. Here, we employ statistical analyses to further characterize the utility of this scoring system. METHODS: A total of 170 nucleated red blood cells isolated from peripheral blood of four women undergoing elective termination of a trisomy 21 male fetus were analyzed. Each of the four scoring system parameters, whose values range from 0 to 3 points, served as an independent variable to determine its significance for the correct identification of a cell as fetal or maternal. A logistic regression was used as a multivariable statistical tool. RESULTS: Forty-four patterns of the four parameters were found in the overall series. Some patterns were exclusively associated with maternal cells (e.g. 1-1-1-1 and 1-1-2-2), and others were exclusively associated with fetal cells (e.g. 3-2-2-2 and 3-2-3-2). A detection rate of 73.9% at a false-positive rate of 5% resulted from a random simulation model performed with a 1:5 case:control matched set. The variables most predictive of a cell being fetal were gamma hemoglobin staining intensity of cytoplasm and nuclear roundness. CONCLUSIONS: The modified scoring system presented here improves upon the previously reported, unmodified erythroblast scoring system. These statistical analyses suggest that the scoring system is a promising method that aids in distinguishing fetal and maternal NRBCs for prenatal diagnostic applications and that it may be amenable to automated microscopy by applying the discrete morphological parameters as computational classifiers.

Cervical cancer and microchimerism.

OBJECTIVE: To determine whether microchimerism is involved in the pathogenesis or progression of cervical cancer. METHODS: Cervical tissue was obtained from eight women who had at least one live-born son and who underwent radical hysterectomy after a diagnosis of cervical cancer. Control tissue was obtained from four women without cervical cancer who had at least one live-born son and from three women with cervical cancer and no male births. Tissue sections were analyzed with fluorescence in situ hybridization for the presence of fetal cells, defined by an X and Y chromosome. Immunolabeling was used to determine the phenotype of the presumed fetal cells. RESULTS: Male cells were found in cervical tissue from all four patients for whom large sections (approximately 1.5 x 2 cm) were analyzed. Only one male cell was found in two of the four patients for whom small biopsy specimens (approximately 0.1 x 0.5 cm) were analyzed. No male cells were found in tissue specimens from controls, whether they were small or large sections. In immunolabeling studies, eight of 18 male cells from one patient were CD45-positive and nine of 37 male cells from two patients were cytokeratin-positive. No cells were positive for both markers. CONCLUSION: Cervical cancer might be associated with microchimerism, possibly from fetomaternal cell trafficking. These results further expand the potential relationship between microchimerism and disease in women.

A simple and sensitive erythroblast scoring system to identify fetal cells in maternal blood.

OBJECTIVES: Nucleated red blood cells (NRBCs) of fetal origin appear to have distinguishable characteristics from that of maternal NRBCs in both nuclear morphology and properties of hemoglobin staining. However, these differences have yet to be quantified. Our aim was to develop an erythroblast scoring system using four distinct phenotypic parameters (nuclear roundness, nuclear morphology, gamma hemoglobin staining intensity, and peripheral brightness of the stained cytoplasm) to address this issue. METHODS: NRBCs were isolated from four maternal blood samples by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection after elective termination of a trisomy 21 male fetus (47,XY,+21). All cells were deposited onto microscope slides and every NRBC was analyzed according to the scoring system. Each of the four individual parameters was given a value from 0 to 3 points and a combined score was obtained for each cell (range 0-12). Fluorescence in situ hybridization (FISH) using X- and Y-specific probes was performed to determine, on the basis of interphase karyotype, whether the cell was maternal or fetal. RESULTS: The majority of maternal NRBCs were found to have a combined score of 6 or less (103/117) and the majority of fetal NRBCs were found to have a score of 7 or greater (43/53). The proportion of cells that were identified correctly as fetal increased with each ascending category of combined score. For example, 5.7% of NRBCs with a combined score of 5 points or less were found to be fetal, whereas 19.2% of NRBCs with a combined score of 6 points were fetal. At combined scores of 11 and 12 points, 100% of NRBCs were found to be fetal. CONCLUSION: Fetal NRBCs have characteristic morphology and a gamma hemoglobin staining appearance that makes them distinguishable from maternal NRBCs. The scoring system presented here is a simple and sensitive method to distinguish fetal NRBCs from adult cells in maternal blood. This system may have clinical utility for noninvasive prenatal diagnosis as well as applications for basic research into the developmental biology of NRBCs. In addition, these defined parameters may serve as computational classifiers for the automated detection of fetal cells in maternal blood.