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Aim: To predict the prognosis of infants with congenital heart disease, accurate prenatal diagnosis of structural abnormality and heart failure are both necessary. The aim of this study was to investigate whether cardiovascular profile (CVP) and biophysical profile (BP) scores are useful for predicting prognosis in infants with congenital heart defect (CHD)., Methods: A retrospective review of singletons prenatally diagnosed with CHD at a tertiary pediatric cardiac center between 2011 and 2015 was undertaken., Results: A total of 202 patients with CHD were analyzed. Perinatal and infant deaths occurred in 16 (7.9%) and 10 cases (5.0%), respectively. Infants with the last CVP score ≤ 5 had 18.7-fold higher perinatal mortality than those with a last CVP score > 5 (P < 0.01). Infants with a last BP score ≤ 6 had 18.7-fold higher perinatal mortality than those with a last BP score > 6 (P < 0.01). Infants with a CVP score decrease in utero had 4.5-fold higher infant mortality than those with an increase or no change (P < 0.01). Multivariate analysis showed that single-ventricle physiology, pre-term birth at <37 weeks of gestation, last CVP score ≤ 5, and last BP score ≤ 6 were independent predictors of perinatal mortality. Single-ventricle physiology and a CVP score decrease were independent predictors of infant mortality., Conclusion: CVP and BP scores are useful for predicting perinatal prognosis in infants with CHD. A CVP score decrease in utero is associated with infant mortality, suggesting that serial CVP score assessment may be useful for management planning., (© 2019 Japan Society of Obstetrics and Gynecology.)

Objective: This study aimed to evaluate the impact of obesity on early evaluation of fetal cardiac landmarks using a standardized examination method at the time of nuchal translucency scan., Methods: This was a cross-sectional study of an ongoing prospective cohort at high risk for congenital heart defects. We used a standardized examination protocol using 2-dimensional sonography with power Doppler in the evaluation of fetal cardiac landmarks consisting of 4-chamber view, outflow tract relationship, and transverse arches view. The study population was stratified based on maternal body mass index into nonobese (<30 kg/m 2 ) and obese (≥30 kg/m 2 ). Groups were compared in terms of satisfactory evaluation of fetal cardiac landmarks, transvaginal sonography use, and scan times required for the evaluations. Subanalysis was performed by further categorizing obesity into nonmorbid obesity (30.0-39.9 kg/m 2 ) and morbid obesity (≥40 kg/m 2 )., Results: A total of 190 patients were evaluated. Of these, 48.4% (n = 92) were obese. The most common indication for fetal cardiac assessment was maternal pregestational diabetes mellitus (42.6%). Transvaginal sonography was utilized in one nonobese woman (1.4%) and 11 obese women (12%) (P = .002). The satisfactory evaluation of 4-chamber view, outflow tract relationship, transverse arches view, and all views were not significantly different between groups (P > .05). The scan time was about 5 minutes longer in the obese group compared with nonobese group (P = .020)., Conclusions: Obesity does not hamper early evaluation of fetal cardiac landmarks around the time of nuchal translucency scan. However, obese patients are more likely to require transvaginal examinations., (© 2018 by the American Institute of Ultrasound in Medicine.)

Objective: Annular plane systolic excursion (APSE) has been evaluated for the right (RV) and left (LV) ventricles using M-mode echocardiography. This study examined APSE using 2-dimensional speckle tracking (2DST) of the lateral and septal annuli of the RV and LV in normal fetuses., Methods: Two hundred normal fetuses were prospectively examined between 20 and 40 weeks' gestation, in which the end-diastolic and end-systolic lengths were measured from the apex to the insertion of the annuli into the lateral and septal walls of the RV and LV using 2DST. Subtracting the end-systolic from the end-diastolic length resulted in the APSE measurement. The APSE values from the ventricular and septal locations were regressed against biometric and gestational age independent variables, and the mean and standard deviation computed using fractional polynomial analysis. Within-subject repeated measures of variance were used to compare results within and between the right ventricular and left ventricular basal APSE values., Results: When regressed against the independent variables the R 2 for the APSE of the right ventricular lateral wall ranged from 0.39 to 0.40, the left ventricular lateral wall 0.29 to 0.31, the right ventricular septal wall 0.22 to 0.40, and the left ventricular septal wall 0.05 to 0.07. There was a significant difference (P < .001) for APSE between the right ventricular and left ventricular lateral wall and their respective septal annuli, the left ventricular and right ventricular lateral wall annuli, and the left ventricular and right ventricular septal annuli., Conclusions: The right ventricular and left ventricular lateral and septal wall APSE can be computed using 2DST and are associated with changing fetal biometric and age measurements., (© 2018 by the American Institute of Ultrasound in Medicine.)

Objectives: The association of an abnormal fetal heart rate (FHR) and chromosomal anomalies in the first trimester of pregnancy remains unclear, probably because of the lack of control for known confounding factors. This study was designed to determine whether an increased FHR is an independent risk factor for chromosomal anomalies between 11 and 14 weeks' gestation., Methods: This cohort study included women who underwent first-trimester genetic screening between 2011 and 2014 at a single institution. A multivariable logistic regression analysis was performed to determine whether an FHR of 170 beats per minute (bpm) or higher, derived from a receiver operating characteristic curve, is an independent risk factor for all chromosomal anomalies while controlling for known confounding factors. P < .05 was considered significant., Results: An FHR of 170 bpm or higher was observed in 7% (228 of 3254), and chromosomal anomalies were present in 1.0% (31 of 3254) of the population. A higher proportion of fetuses with an FHR of 170 bpm or higher had chromosomal anomalies compared to those with an FHR lower than 170 bpm. An FHR of 170 bpm or higher was an independent risk factor for chromosomal anomalies after controlling for known confounding factors. Of note, in the group of fetuses with a nuchal translucency above the 95th percentile, the frequency of chromosomal anomalies was significantly higher among fetuses with an FHR of 170 bpm or higher compared to those with an FHR lower than 170 bpm., Conclusions: Fetal tachycardia is a risk factor for chromosomal anomalies during first-trimester genetic screening, independent of increased nuchal translucency, nuchal septations, and maternal age., (© 2018 by the American Institute of Ultrasound in Medicine.)

Introduction: This study was conducted to evaluate the 24-segment transverse widths of the right and left ventricles distributed from the base to the apex of the ventricular chambers and compute the right ventricular (RV)/left ventricular (LV) ratios for each segment., Methods: Two hundred control fetuses were examined between 20 and 40 weeks' gestation. Using offline speckle-tracking software, the 24 end-diastolic transverse widths were computed and the RV/LV ratios were regressed against 7 independent variables related to the size, weight, and age of the fetuses. Five fetuses with coarctation of the aorta and 5 fetuses with pulmonary stenosis were examined to exemplify the utility of these measurements., Findings: The 24-segment transverse widths were associated with changes in fetal size, weight, and age. Regression equations were developed to describe these changes with R 2 values between .5 and .82. The measurements were normally distributed (Shapiro-Wilk > 0.5). The RV/LV ratio for the 24 segments did not strongly correlate (R 2  = .001 to -.2) with fetal size, weight, or gestational age. Fetuses with coarctation of the aorta and pulmonary stenosis demonstrated characteristic changes in the 24-segment transverse widths and the RV/LV ratios in the basal (segments 1-8), mid (segments 9-16) and apical (segments 17-24) sections of the ventricles., Conclusions: The 24-segment transverse widths of the right and left ventricles and the RV/LV ratios provide a comprehensive method to examine the width of the ventricular chambers., (© 2018 by the American Institute of Ultrasound in Medicine.)

Ventricular Septal Defect (VSD), the most common congenital heart defect, is characterized by a hole in the septum between the right and left ventricles. The pathogenesis of VSD is unknown in most clinical cases. There is a paucity of data relevant to epigenetic changes in VSD. The placenta is a fetal tissue crucial in cardiac development and a potentially useful surrogate for evaluating the development of heart tissue. To understand epigenetic mechanisms that may play a role in the development of VSD, genome-wide DNA methylation assay on placentas of 8 term subjects with isolated VSD and no known or suspected genetic syndromes and 10 unaffected controls was performed using the Illumina HumanMethylation450 BeadChip assay. We identified a total of 80 highly accurate potential CpGs in 80 genes for detection of VSD; area under the receiver operating characteristic curve (AUC ROC) 1.0 with significant 95% CI (FDR) p-values < 0.05 for each individual locus. The biological processes and functions for many of these differentially methylated genes are previously known to be associated with heart development or disease, including cardiac ventricle development (HEY2, ISL1), heart looping (SRF), cardiac muscle cell differentiation (ACTC1, HEY2), cardiac septum development (ISL1), heart morphogenesis (SRF, HEY2, ISL1, HEYL), Notch signaling pathway (HEY2, HEYL), cardiac chamber development (ISL1), and cardiac muscle tissue development (ACTC1, ISL1). In addition, we identified 8 microRNAs that have the potential to be biomarkers for the detection of VSD including: miR-191, miR-548F1, miR-148A, miR-423, miR-92B, miR-611, miR-2110, and miR-548H4. To our knowledge this is the first report in which placental analysis has been used for determining the pathogenesis of and predicting VSD., Competing Interests: The authors have declared that no competing interests exist.

This study evaluates the impact of extended cardiac views on examination time, repeat imaging, and anomaly detection before and after implementation of 76811 guidelines (American Institute of Ultrasound in Medicine Consensus 2014). It is a retrospective study of singleton pregnancies undergoing detailed ultrasound imaging at 18 weeks' gestation or greater before and after the protocol change in an academic, tertiary care fetal center. Views required prior to 2014: 4-chamber, left outflow tract, right outflow tract. Additional views required after 2014: bicaval, aortic arch, 3-vessel, and 3-vessel trachea. Fetuses with known anomalies were excluded. Rates of detection of congenital heart disease (CHD), examination completion, repeat examination recommendation, fetal echocardiogram recommendation, completion by body mass index, and cardiac examination time were determined. Six hundred twenty-four subjects were included, 217 before and 407 after protocol change. Views obtained were as stated in the American Institute of Ultrasound in Medicine/Society for Maternal-Fetal Medicine consensus. Detection of CHD was not improved. Examination times increased by 20% (6.4 vs 7.7 minutes, P < 0.05). Number of incomplete studies increased by 130% (11% to 26%, P < 0.05). Twice as many patients were referred for repeat examination (6% vs 13%, P < 0.05). Completion rates were negatively correlated with body mass index. Recommendations for fetal echocardiogram were unchanged (5% vs 6%, P = 0.6). Additional imaging did not increase detection rate of CHD (3% vs 2%, P = 0.3). Extended cardiac views resulted in increased examination time, more incomplete examinations, and more repeat examinations without changing detection rates of CHD.

The aims of this study were to demonstrate the feasibility of obtaining additional cardiac views as proposed on an extended fetal cardiac examination and to see if there was any variation in individual components of that examination stratified by sonographer training, patient body habitus, or equipment. We retrospectively reviewed 200 consecutive detailed second-trimester high-risk fetal obstetric sonograms that included additional extended cardiac views. We analyzed the percentage of the time individual views were obtained, with variation based on (1) a sonographer with greater than 3 years of training compared with a group with 6 to 12 months of training, (2) 2 different ultrasound units, and (3) different body mass indices. Overall, the highest rate of visualization was achieved with the 4-chamber view (98.2%), whereas the 3-vessel tracheal view had the lowest percentage of visualization (40.2%), among the less experienced sonographers. Differences in successful completion of the extended cardiac views were not statistically different between the sonographer with a level of training greater than 3 years as compared with those with 6 to 12 months' training except for the 3-vessel tracheal view (P < 0.001). There is no statistically significant difference in our ultrasound equipment, when considering only inexperienced sonographers. Increasing body mass index had an inverse relationship with obtaining the components of the detailed cardiac examination. Using state-of-the-art ultrasound equipment and with focused additional training of obstetric sonographers, the majority of extended cardiac views can be obtained. There are exceptions.

Congenital cardiac anomalies are a common finding during prenatal anatomical survey ultrasound examination. Cardiac anomalies are a major cause of prenatal and neonatal mortality and morbidity. If the anomaly is not lethal, most would require surgical correction. Therefore, early recognition of these abnormalities is essential for parental counseling and delivery planning, as well as analysis of neonatal treatment options. Although prenatal ultrasound plays an important role in identification of such anomalies, diagnosis and interpretation of imaging findings require familiarity and knowledge of the common imaging features. In this article, we provide a comprehensive review of ultrasound appearance of common fetal cardiac anomalies.

Objective: We attempted to evaluate fetal cardiac activity of congenital heart disease (CHD) and normal heart fetuses by magnetic resonance imaging (MRI)., Methods: We evaluated the fetal cardiac functional assessment and the blood flow of descending aorta at 34 weeks' gestation or more by MRI and compared the results with ultrasonography findings. We measured 6 normal heart fetuses and 14 CHD fetuses., Results: The ejection fraction (EF) and descending aorta blood flow in the CHD group were 76.4% ± 11.9%, 687.5 ± 303.8 mL/min by ultrasonography and 48.3% ± 7.1%, 711.4 ± 273.1 mL/min by MRI; those in the normal group were 66.9% ± 12.2%, 898.1 ± 245.9 mL/min by ultrasonography and 51.3% ± 4.2%, 911.9 ± 223.1 mL/min by MRI., Conclusions: There was no significant difference in descending aorta blood flow measurements between ultrasonography and MRI, but the EF showed a significant between-group difference. In the normal group, the variation of EF and blood flow measured by MRI was significantly smaller compared with those of ultrasonography.

Congenital heart disease (CHD) is the most common type of birth defect and is both a significant pediatric and adult health problem, in light of a growing population of survivors. The etiology of CHD has been considered to be multifactorial with genetic and environmental factors playing important roles. The combination of advances in cardiac developmental biology, which have resulted in the elucidation of molecular pathways regulating normal cardiac morphogenesis, and genome sequencing technology have allowed the discovery of numerous genetic contributors of CHD ranging from chromosomal abnormalities to single gene variants. Conversely, mechanistic details of the contribution of environmental factors to CHD remain unknown. Maternal diabetes mellitus (matDM) is a well-established and increasingly prevalent environmental risk factor for CHD, but the underlying etiologic mechanisms by which pregestational matDM increases the vulnerability of embryos to cardiac malformations remains largely elusive. Here, we will briefly discuss the multifactorial etiology of CHD with a focus on the epidemiologic link between matDM and CHD. We will describe the animal models used to study the underlying mechanisms between matDM and CHD and review the numerous cellular and molecular pathways affected by maternal hyperglycemia in the developing heart. Last, we discuss how this increased understanding may open the door for the development of novel prevention strategies to reduce the incidence of CHD in this high-risk population., (© 2018 Wiley Periodicals, Inc.)

Background: The objective of this study was to investigate the expression and localisation of thymosin β4 (Tβ4) in the developing human heart. Tβ4 is a cardioprotective protein which may have therapeutic potential. While Tβ4 is an endogenously produced protein with known importance during development, its role within the developing human heart is not fully understood. Elucidating the localisation of Tβ4 within the developing heart will help in understanding its role during cardiac development and is crucial for understanding its potential for cardioprotection and repair in the adult heart., Methods: Expression of Tβ4 mRNA in the early fetal human heart was assessed by PCR using both ventricular and atrial tissue. Fluorescence immunohistochemistry was used to assess the localisation of Tβ4 in sections of early fetal human heart. Co-staining with CD31, an endothelial cell marker, and with myosin heavy chain, a cardiomyocyte marker, was used to determine whether Tβ4 is localised to these cell types within the early fetal human heart., Results: Tβ4 mRNA was found to be expressed in both the atria and the ventricles of the early fetal human heart. Tβ4 protein was found to be primarily localised to CD31-expressing endothelial cells and the endocardium as well as being present in the epicardium. Tβ4-associated fluorescence was greater in the compact layer of the myocardial wall and the interventricular septum than in the trabecular layer of the myocardium., Conclusions: The data presented illustrates expression of Tβ4 in the developing human heart and demonstrates for the first time that Tβ4 in the human heart is primarily localised to endothelial cells of the cardiac microvasculature and coronary vessels as-well as to the endothelial-like cells of the endocardium and to the epicardium., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Fetal endoscopic tracheal occlusion (FETO) is offered to fetuses with congenital diaphragmatic hernia (CDH) and severe lung hypoplasia to promote lung growth and may secondarily affect left heart growth. The effects of FETO on left heart hypoplasia (LHH) are not described post-CDH repair., Methods: A retrospective analysis was performed for fetuses with left-sided CDH who underwent FETO and severity-matched controls from 2007 to 2016 at our institution. Echocardiographic, ultrasound, and MRI data were reviewed. Left heart dimensions were assessed prenatally and postnatally. Primary clinical outcome evaluated was death., Results: Twelve FETO patients and 18 controls were identified. Fetal LHH was noted in both groups and worsened after FETO. Postnatal mitral valve dimensions were larger in the FETO group pre-CDH repair (P = .03). Post-CDH repair, mitral valve and left ventricular dimensions were not significantly different between groups (P = .79 and P = .63 respectively) while FETO aortic valve dimensions were smaller (P = .04). Extracorporeal membrane oxygenation use was lower in the FETO group. No associations were found between left heart dimensions and outcomes., Conclusion: Although increased lung growth was seen after FETO, fetal LHH persisted with relative normalization seen post-repair. Persistent LHH post-FETO could be secondary to a small contribution of pulmonary venous return to the fetal left heart and increased intrathoracic pressures post-FETO., (© 2018 John Wiley & Sons, Ltd.)

Background: Valproic acid (VPA) exposure during pregnancy has been proven to contribute to congenital heart disease (CHD). Our previous findings implied that disruption of planar cell polarity (PCP) signaling pathway in cardiomyocytes might be a factor for the cardiac teratogenesis of VPA. In addition, the teratogenic ability of VPA is positively correlated to its histone deacetylase (HDAC) inhibition activity. This study aimed to investigate the effect of the VPA on cardiac morphogenesis, HDAC1/2/3, and PCP key genes (Vangl2/Scrib/Rac1), subsequently screening out the specific HDACs regulating PCP pathway., Methods: VPA was administered to pregnant C57BL mice at 700 mg/kg intraperitoneally on embryonic day 10.5. Dams were sacrificed on E15.5, and death/absorption rates of embryos were evaluated. Embryonic hearts were observed by hematoxylin-eosin staining to identify cardiac abnormalities. H9C2 cells (undifferentiated rat cardiomyoblasts) were transfected with Hdac1/2/3 specific small interfering RNA (siRNA). Based on the results of siRNA transfection, cells were transfected with Hdac3 expression plasmid and subsequently mock-treated or treated with 8.0 mmol/L VPA. Hdac1/2/3 as well as Vangl2/Scrib/Rac1 mRNA and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. Total HDAC activity was detected by colorimetric assay., Results: VPA could induce CHD (P < 0.001) and inhibit mRNA or protein expression of Hdac1/2/3 as well as Vangl2/Scrib in fetal hearts, in association with total Hdac activity repression (all P < 0.05). In vitro, Hdac3 inhibition could significantly decrease Vangl2/Scrib expression (P < 0.01), while knockdown of Hdac1/2 had no influence (P > 0.05); VPA exposure dramatically decreased the expression of Vanlg2/Scrib together with Hdac activity (P < 0.01), while overexpression of Hdac3 could rescue the VPA-induced inhibition (P > 0.05)., Conclusion: VPA could inhibit Hdac1/2/3, Vangl2/Scrib, or total Hdac activity both in vitro and in vivo and Hdac3 might participate in the process of VPA-induced cardiac developmental anomalies., Competing Interests: There are no conflicts of interest

Background: Fetal echocardiography can accurately diagnose critical congenital heart disease prenatally, but relies on referrals from abnormalities identified on routine obstetrical ultrasounds. Critical congenital heart disease that is frequently missed due to inadequate outflow tract imaging includes anomalies such as truncus arteriosus, double outlet right ventricle, transposition of the great arteries, tetralogy of Fallot, pulmonary stenosis, and aortic stenosis., Objective: This study evaluated the prenatal detection rate of critical outflow tract anomalies in a single urban pediatric hospital before and after "AIUM Practice Guideline for the Performance of Obstetric Ultrasound Examinations," which incorporated outflow tract imaging., Design: Infants with outflow tract anomalies who required cardiac catheterization and/or surgical procedure(s) in the first 3 months of life were retrospectively identified. This study evaluated two time periods; pre-guidelines from June 2010 to May 2013 and post-guidelines from January 2015 to June 2016. June 2013-December 2014 was excluded as a theoretical period necessary for obstetrical practices to implement the revised guidelines., Results: Overall, prenatal diagnosis occurred in 55% of infants with critical outflow tract anomalies; of the three most common defects, prenatal diagnosis occurred in 53% of D-transposition of the great arteries, 63% of tetralogy of Fallot, and 80% of double outlet right ventricle patients. Pre-guidelines, prenatal diagnosis occurred in 52% (52 of 102) infants with critical outflow tract anomalies requiring early cardiac intervention. Post-guidelines, prenatal diagnosis occurred in 61% (33 of 54) infants, not significantly different than the prenatal detection rate pre-guidelines (P = .31)., Conclusions: Despite revised obstetrical guidelines highlighting the importance of outflow tract imaging, referrals and prenatal diagnosis of these types of critical congenital heart disease remain low. Education of obstetrical sonographers and practitioners who perform fetal anatomic screening is vital to increase referrals and prenatal detection of critical outflow tract anomalies., (© 2018 Wiley Periodicals, Inc.)

TSP50, a testis-specific gene encoding a serine protease-like protein, was specifically expressed in the spermatocytes of testes but abnormally activated and expressed in many different kinds of cancers. Here, we aimed to analyze the expression of TSP50 in mouse embryo and its function in early embryonic development. Firstly, the distribution of TSP50 in oocytes and embryonic development was characterized by immunofluorescence, RT-PCR and western blotting, and the results showed that TSP50 was detected at all studied stages with a dynamic expression pattern. When overexpressed TSP50 in zygotes by microinjection, the zygotes development was highly accelerated. On the contrary, knocking down TSP50 expression by RNA interference greatly retarded the zygote development. Furthermore, TSP50 expression at embryonic day 6.5 (E6.5), day 8.5 (E8.5) and day 10.5 (E10.5) were increasingly enhanced, However, the expression of TSP50 decreased gradually in the development and differentiation of cardiac myocyte from E12.5 to postnatal (P0). Additionally, we found that TSP50 expression was decreased during cardiac myocyte differentiation of P19 cells. Overexpression of TSP50 could decrease the expression of GATA-4, and knockdown of TSP50 markedly increase the expression of GATA-4. Taken together, our data indicate that TSP50 may play an important role during the process of mouse embryonic development as well as myocardial cell differentiation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Placental insufficiency, high altitude pregnancies, maternal obesity/diabetes, maternal undernutrition and stress can result in a poor setting for growth of the developing fetus. These adverse intrauterine environments result in physiological changes to the developing heart that impact how the heart will function in postnatal life. The intrauterine environment plays a key role in the complex interplay between genes and the epigenetic mechanisms that regulate their expression. In this review we describe how an adverse intrauterine environment can influence the expression of miRNAs (a sub-set of non-coding RNAs) and how these changes may impact heart development. Potential consequences of altered miRNA expression in the fetal heart include; Hypoxia inducible factor (HIF) activation, dysregulation of angiogenesis, mitochondrial abnormalities and altered glucose and fatty acid transport/metabolism. It is important to understand how miRNAs are altered in these adverse environments to identify key pathways that can be targeted using miRNA mimics or inhibitors to condition an improved developmental response., Competing Interests: The authors declare no conflict of interest.

Objectives: Although the postnatal physiology of D-loop transposition of the great arteries with intact ventricular septum (D-TGA/IVS) is well established, little is known about fetal D-TGA/IVS. In the normal fetus, the pulmonary valve (PV) is larger than the aortic valve (AoV), there is exclusive right-to-left flow at the foramen ovale (FO) and ductus arteriosus (DA), and the left ventricle (LV) ejects 40% of combined ventricular output (CVO) through the aorta, primarily to the brain. In D-TGA/IVS, the LV ejects oxygen-rich blood to the pulmonary artery, theoretically leading to pulmonary vasodilation, increased branch pulmonary artery flow and reduced DA flow. In this study, we tested the hypothesis that D-TGA/IVS anatomy results in altered cardiac valve sizes, ventricular contribution to CVO, and FO and DA flow direction., Methods: Seventy-four fetuses with D-TGA/IVS that underwent fetal echocardiography at our institution between 2004 and 2015 were included in the study. AoV, PV, mitral valve and tricuspid valve sizes were measured and Z-scores indexed to gestational age were generated. Ventricular output was calculated using Doppler-derived velocity-time integral, and direction of flow at the FO and DA shunts was recorded in each fetus using both color Doppler and flap direction. Measurements in the D-TGA/IVS fetuses were compared with data of 222 controls, matched for gestational-age range, from our institutional normal fetal database., Results: The LV component of CVO was higher in D-TGA/IVS fetuses than in controls (50.7% vs 40.2%; P < 0.0001), with no difference in the total CVO. Flow was bidirectional at the FO in 56 (75.7%) and at the DA in 24 (32.4%) D-TGA/IVS fetuses. Only 21.6% fetuses had normal right-to-left flow at both shunts. Bidirectional shunting was more common in third-trimester fetuses than in second-trimester ones (P < 0.03). AoV and PV diameters were nearly identical in D-TGA/IVS in contrast to control fetuses, hence AoV Z-score was higher than PV Z-score (1.13 vs -0.65, P < 0.0001) in D-TGA/IVS., Conclusions: In fetuses with D-TGA/IVS there is loss of the normal right-sided dominance, as each ventricle provides half of the CVO, with a relatively large AoV diameter and a small PV diameter, and high incidence of bidirectional FO and DA flow. This may support the theory that high pulmonary artery oxygen content reduces pulmonary vascular resistance, thereby increasing branch pulmonary artery flow and venous return, which results in increased LV preload and output. Pulmonary sensitivity to oxygen is thought to increase later in gestation, which may explain the higher incidence of bidirectional shunting. Consequences of these flow alterations include increased aortic and, most likely, brain flow, perhaps in an attempt to compensate for the substrate deficiency observed in D-TGA/IVS. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd., (Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.)

The developmental timeline of the human heart remains elusive. The heart takes on its characteristic four chambered appearance by ~56 days gestational age (DGA). However, owing to the complexities (both technical and logistical) of exploring development in utero, we understand little of how the ventricular walls develop. To address this, we employed diffusion tensor magnetic resonance imaging to explore the architecture and tissue organization of the developing heart aged 95-143 DGA. We show that fractional anisotropy increases (from ~0.1 to ~0.5), diffusion coefficients decrease (from ~1 × 10 -3 mm 2 /sec to ~0.4 × 10 -3 mm 2 /sec), and fiber paths, extracted by tractography, increase linearly with gestation, indicative of the increasing organization of the ventricular myocytes. By 143 DGA, the developing heart has the classical helical organization observed in mature mammalian tissue. This was accompanied by an increase in connexin 43 and connexin 40 expression levels, suggesting their role in the development of the ventricular conduction system and that electrical propagation across the heart is facilitated in later gestation. Our findings highlight a key developmental window for the structural organization of the fetal heart.

Background: Early fetal echocardiography (FE), performed at 12 to 16 weeks' gestational age (GA), can be used to screen for fetal heart disease akin to that routinely performed in the second trimester. The efficacy of FE at earlier GAs has not been as well explored, particularly with recent advances in ultrasound technology. The aim of this study was to evaluate the efficacy of early FE in assessing fetal heart structure, and the added benefit of color Doppler (CD), from as early as 6 weeks through to 13 +6  weeks' GA., Methods: Pregnant women were prospectively recruited for first-trimester FE. All underwent two-dimensional (2D) cardiac imaging combined with CD assessment, and all were offered second-trimester fetal echocardiographic evaluations. Fetal cardiac anatomy was assessed both in real time during FE and additionally offline by two separate reviewers., Results: Very early FE was performed in 202 pregnancies including a total of 261 fetuses, with 92% (n = 241) being reassessed at ≥18 weeks' GA. Mean GA at FE was 10 +6  weeks (range, 6 +1 to 13 +6  weeks). Transabdominal scanning was used in all cases, and transvaginal scanning was used additionally in most at <11 weeks' GA (n = 103 of 117 [88%]). There was stepwise improvement in image resolution of the fetal heart in those pregnancies that presented at later gestation for assessment. CD assisted with definition of cardiac anatomy at all GAs. A four-chambered heart could be identified in 52% of patients in the eighth week (n = 12 of 23), improving to 80% (n = 36 of 45) in the 10th week and 98% (n = 57 of 58) by the 11th week. The inferior vena cava was visualized by 2D imaging in only 4% (n = 1 of 23) in the eighth week, increasing to 13% (n = 6 of 45) by the 10th week and 80% (n = 25 of 31) by the 13th week. CD improved visualization of the inferior vena cava at earlier GAs to >80% (n = 37 of 45) from 10 weeks. Pulmonary veins were not visualized by either 2D imaging or CD until after the 11th week. Both cardiac outflow tracts could be visualized by 2D imaging in the minority from 8 +0 to 10 +6  weeks (n = 18 of 109 [16%]) but were imaged in most from 11 +0 to 13 +6  weeks (n = 114 of 144 [79%]). CD imaging improved visualization of both outflow tracts to 64% (n = 29 of 45) in the 10th week. On 2D imaging alone, both the aortic and ductal arches were seen in only 29% of patients in the 10th week (n = 13 of 45), increasing to 58% when CD was used (58% [n = 26 of 45]) and to >80% (n = 47 of 58) using CD in the 11th week., Conclusions: Very early FE, from as early as 8 weeks, can be used to assess cardiac structures. The ability to image fetal heart structures between 6 and 8 weeks is currently nondiagnostic. The use of CD significantly increases the detection of cardiac structures on early FE. The ideal timing of complete early FE, excluding pulmonary vein assessment, appears to be after 11 weeks' GA., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

Background: The authors investigated left ventricular (LV) rotational indices, twist, and torsion in a large cohort of fetuses using two-dimensional speckle-tracking echocardiography., Methods: Pregnant women (N = 102) were prospectively recruited for fetal two-dimensional speckle-tracking echocardiography. Because of variable fetal position, ventricular orientation was established using the location of the liver and LV position in relation to the right ventricle. Twist measurements required correction to account for fetal position, reconciling directionality across all fetal orientations. Peak apical and basal rotations, global longitudinal strain, global circumferential strain (GCS), longitudinal strain rate, circumferential strain rate (CSR), twist, and torsion were reported and tested for associations with gestational age (GA) and estimated fetal weight (EFW)., Results: Measurement of rotational indices was feasible in 175 examinations (73%). The mean maternal age was 31 ± 6 years, the mean GA 24 ± 6 weeks, and the EFW 1.0 ± 1.0 kg. Mean peak apical rotation, basal rotation, twist, and torsion were 9.5 ± 2.0°, -4.4 ± 1.1°, 13.1 ± 2.3°, and 7.9 ± 3.4°/cm, respectively. Mean global longitudinal strain, GCS, longitudinal strain rate, and CSR were -22.3 ± 4.3%, -25.0 ± 6.1%, -1.4 ± 0.5 sec -1 , and -1.6 ± 0.5 sec -1 , respectively. Absolute GCS, CSR, apical rotation, and twist declined moderately with GA and EFW (P < .05). There were strong negative nonlinear correlations of torsion with GA and EFW: torsion = 1,104 × GA -1.591 (r 2  = 0.760, P < .001); torsion = 115.88 × EFW -0.427 (r 2  = 0.772, P < .001)., Conclusions: Determination of fetal LV rotational mechanics is feasible and reproducible using two-dimensional speckle-tracking echocardiography. Basal rotation, longitudinal strain, and strain rate vary little with EFW and GA. Interpretation of LV apical rotation, twist, torsion, GCS, and CSR, however, must take GA or EFW into account. These data form the basis for comparison with findings in fetuses with abnormal cardiac anatomy and function., (Copyright © 2017 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.)

Background: Identification of prenatal ventriculoarterial connections in fetuses with conotruncal anomalies (CTA) remains one of the greatest challenges for sonographers performing screening examinations. Herein, we propose a novel protocol of 4D volume analysis that identifies ventriculoarterial connections and evaluate its clinical utility in routine screenings., Methods: Twenty-nine cases of transposition of the great arteries (TGA), 22 cases of double-outlet right ventricle (DORV), 36 cases of tetralogy of Fallot (TOF), 14 cases of truncus arteriosus (TCA), and randomly selected 70 normal fetuses were reviewed in this study. All cases were evaluated using 2D data alone (2D method), post-processing volumes with no exact algorithm (4D-1 method), or with the proposed algorithm (4D-2 method), or using the 2D and 4D data together (combined method). Comparisons were made to evaluate the detection rate of ventriculoarterial connections for these different methods., Results: During 18-28 gestational weeks, the detection rate of 4D-2 modality was satisfactory. The detection rate of the combined method was significantly higher than 2D method in the identification of TGA, TOF, and TCA. The detection rate of 4D-1 method was significantly lower than 4D -2 modality for CTA fetuses. During late pregnancy, the detection rate for both 4D modalities was very low due to the poor quality of the 4D volumes., Conclusions: We proposed a detailed protocol, which allowed the examiner to identify fetal ventriculoarterial connections by 4D volumes. Inclusion of blood information into the volumes improved diagnosis. Our findings suggest that the incorporation of 4D STIC into routine screenings could improve the detection for TGA, TOF, and TCA.

Although many changes have been discovered during heart maturation, the genetic mechanisms involved in the changes between immature and mature myocardium have only been partially elucidated. Here, gene expression profile changed between the human fetal and adult heart was characterized. A human microarray was applied to define the gene expression signatures of the fetal (13-17 weeks of gestation, n = 4) and adult hearts (30-40 years old, n = 4). Gene ontology analyses, pathway analyses, gene set enrichment analyses, and signal transduction network were performed to predict the function of the differentially expressed genes. Ten mRNAs were confirmed by quantificational real-time polymerase chain reaction. 5547 mRNAs were found to be significantly differentially expressed. "Cell cycle" was the most enriched pathway in the down-regulated genes. EFGR, IGF1R, and ITGB1 play a central role in the regulation of heart development. EGFR, IGF1R, and FGFR2 were the core genes regulating cardiac cell proliferation. The quantificational real-time polymerase chain reaction results were concordant with the microarray data. Our data identified the transcriptional regulation of heart development in the second trimester and the potential regulators that play a prominent role in the regulation of heart development and cardiac cells proliferation.

Fetuses with hypoplastic left heart syndrome (HLHS) and intact atrial septum are a particular subset of HLHS neonates with high perinatal mortality. The reported mortality in these patients is 50% to 70%, even with prenatal diagnosis. Prenatal left atrial and pulmonary venous hypertension results in abnormal pulmonary vascular and parenchymal development. The goal of this study was to compare the sonographic appearance of the lungs in fetuses with HLHS/intact atrial septum to neonatal outcome and/or pathology in cases where in utero intervention was performed to open the atrial septum. We found that lung inhomogeneity on ultrasound corresponded to peripheral lymphatic dilatation at autopsy and was associated with a dismal prognosis even when in utero intervention was successful.

Background: Ventricular noncompaction is characterized by excessive trabeculations and is associated with heart failure. The lesion is hypothesized to result from failed compaction and thus retention of embryonic trabeculations. Here, we assess for the first time the identity of trabeculations in noncompaction to test whether noncompacted hearts show retention of embryonic trabeculations., Methods: Using immunohistochemistry, we analyzed cardiac sections of the heart of a control embryo, 3 cases of fetal noncompaction (a set of twins and an unrelated fetus) and 3 fetal hearts without noncompaction., Results: In the embryo, the ventricular trabeculations strongly expressed ANF/NPPA whereas the compact wall did not. In the noncompaction hearts, trabeculations constituted an excessively thick layer. In noncompaction and control fetal hearts alike, however, only a miniscule subset of sub-endocardial myocardium of the trabeculations most proximal to the central ventricular lumen exhibited strong expression of ANF/NPPA, representing Purkinje myocardium. The trabeculations of both fetal control and noncompaction hearts were ANF-negative and orders of magnitude wider than those of the embryo. Both the compact and noncompaction trabeculated myocardium were rich in coronary vasculature. Like embryonic trabeculations, the ANF + Purkinje myocardium had little if any vasculature., Conclusion: The excessive trabeculations in noncompaction do not have the embryonic identity and noncompaction is probably not the result of failed compaction. We propose the lesion results from the compact wall growing into the ventricular lumen in a trabecular fashion., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Purpose: To construct biventricular reference ranges for isovolumic time intervals (isovolumic contraction time, ICT; isovolumic relaxation time, IRT) and ejection time (ET) for colour tissue Doppler imaging (cTDI) between 15 and 37 weeks' in healthy fetuses., Methods: This was a prospective multicentre cross-sectional study involving 160 singleton pregnancies between 15 and 37 weeks of gestation, using cTDI. ICT, ET, IRT and myocardial performance index (MPI) were analysed offline using a small region of interest (ROI) within the basal part of the right and left ventricular wall immediately distal to the annulus. Regression analysis was used to determine gestational age-adjusted reference ranges and to construct nomograms for cTDI parameters., Results: Right and left ventricular ICT (p = 0.004 and p < 0.001) and ET (p = 0.011 and p = 0.050) increased, whereas IRT (p = 0.862 and p = 0.067) and MPI (p = 0.476 and p = 0.777) remained constant with gestational age., Conclusions: This is the first study to evaluate fetal isovolumic time intervals in the second and third trimesters of gestation using cTDI. Normal data for fetal isovolumic time intervals and biventricular MPI by colour tissue Doppler imaging are provided. The reference ranges may be useful in research or clinical studies and can be used in fetuses with compromised cardiac function.

Diffusion tensor imaging (DTI) has emerged as a promising method for noninvasive quantification of myocardial microstructure. However, the origin and behavior of DTI measurements during myocardial normal development and remodeling remain poorly understood. In this work, conventional and bicompartmental DTI in addition to three-dimensional histological correlation were performed in a sheep model of myocardial development from third trimester to postnatal 5 months of age. Comparing the earliest time points in the third trimester with the postnatal 5 month group, the scalar transverse diffusivities preferentially increased in both left ventricle (LV) and right ventricle (RV): secondary eigenvalues D2 increased by 54% (LV) and 36% (RV), whereas tertiary eigenvalues D3 increased by 85% (LV) and 67% (RV). The longitudinal diffusivity D1 changes were small, which led to a decrease in fractional anisotropy by 41% (LV) and 33% (RV) in 5 month versus fetal hearts. Histological analysis suggested that myocardial development is associated with hyperplasia in the early stages of the third trimester followed by myocyte growth in the later stages up to 5 months of age (increased average myocyte width by 198%, myocyte length by 128%, and decreased nucleus density by 70% between preterm and postnatal 5 month hearts.) In a few histological samples (N = 6), correlations were observed between DTI longitudinal diffusivity and myocyte length (r = 0.86, P < 0.05), and transverse diffusivity and myocyte width (r = 0.96, P < 0.01). Linear regression analysis showed that transverse diffusivities are more affected by changes in myocyte size and nucleus density changes than longitudinal diffusivities, which is consistent with predictions of classical models of diffusion in porous media. Furthermore, primary and secondary DTI eigenvectors during development changed significantly. Collectively, the findings demonstrate a role for DTI to monitor and quantify myocardial development, and potentially cardiac disease. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Congenital double aortic arch (DAA) is an uncommon vascular anomaly; however, its prenatal detection is associated with congenital heart defects and chromosomal abnormalities, including 22q11 deletion. We present a case of DAA diagnosed prenatally. DAA can be diagnosed by prenatal ultrasound in the transverse three vessel-trachea view, which shows a trident image formed by a complete vascular ring and the ductus arteriosus. Postnatal magnetic resonance images in this view correlate well with prenatal ultrasound images and help in confirmation of diagnosis, evaluation of the risk of airway or esophageal compression, and planning of surgery.

Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects. Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: To demonstrate the blood flow profiles of fetuses with cardiac anomalies at the level of Ductus venosus (DV) and Aortic isthmus (AI) to evaluate the effects of fetal cardiac anomalies on these profiles, and how these profile changes contribute to cardiac anomaly screening studies as a marker., Methods: DV and AI doppler studies were applied to 64 singleton pregnant women with fetal cardiac anomalies and 74 pregnant women with healthy fetuses. DV-PVIV (peak velocity index for veins) for DV and IFI (isthmic flow index) for AI were used., Results: DV doppler studies in fetuses with cardiac anomalies and healthy fetuses did not show statistically significant difference. But the results of the AI doppler studies had statistically significant difference in the fetal cardiac anomaly group with the exception of cases with dilatation and regurgitation. When right-sided heart anomaly and the remaining cases were compared with the control groups, AI doppler results also showed lower IFI values., Conclusions: DV doppler studies in the second or third trimester may not be suitable as a screening test for congenital heart disease, but AI doppler studies might be considered as a supporting parameter. But further studies are needed for routine clinical use.

The human fetal heart is formed early during embryogenesis as a result of cell migrations, differentiation, and formative blood flow. It begins to beat around gestation day 22. Progenitor cells are derived from mesoderm (endocardium and myocardium), proepicardium (epicardium and coronary vessels), and neural crest (heart valves, outflow tract septation, and parasympathetic innervation). A variety of molecular disturbances in the factors regulating the specification and differentiation of these cells can cause congenital heart disease. This review explores the contribution of different cardiac progenitors to the embryonic heart development; the pathways and transcription factors guiding their expansion, migration, and functional differentiation; and the endogenous regenerative capacity of the adult heart including the plasticity of cardiomyocytes. Unfolding these mechanisms will become the basis for understanding the dynamics of specific congenital heart disease as well as a means to develop therapy for fetal as well as postnatal cardiac defects and heart failure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Key Points: The contractile properties of human fetal cardiac muscle have not been previously studied. Small-scale approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well-controlled conditions. We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59-134 days. Human fetal cardiac myofibrils have low force and slow kinetics of activation and relaxation that increase during the time period studied, and kinetic changes may result from structural maturation and changes in protein isoform expression. Understanding the time course of human fetal cardiac muscle structure and contractile maturation can provide a framework to study development of contractile dysfunction with disease and evaluate the maturation state of cultured stem cell-derived cardiomyocytes., Abstract: Little is known about the contractile properties of human fetal cardiac muscle during development. Understanding these contractile properties, and how they change throughout development, can provide valuable insight into human heart development, and provide a framework to study the early stages of cardiac diseases that develop in utero. We characterized the contractile properties of isolated human fetal cardiac myofibrils across 8-19 weeks of gestation. Mechanical measurements revealed that in early stages of gestation there is low specific force and slow rates of force development and relaxation, with increases in force and the rates of activation and relaxation as gestation progresses. The duration and slope of the initial, slow phase of relaxation, related to myosin detachment and thin filament deactivation rates, decreased with gestation age. F-actin sliding on human fetal cardiac myosin-coated surfaces slowed significantly from 108 to 130 days of gestation. Electron micrographs showed human fetal muscle myofibrils elongate and widen with age, but features such as the M-line and Z-band are apparent even as early as day 52. Protein isoform analysis revealed that β-myosin is predominantly expressed even at the earliest time point studied, but there is a progressive increase in expression of cardiac troponin I (TnI), with a concurrent decrease in slow skeletal TnI. Together, our results suggest that cardiac myofibril force production and kinetics of activation and relaxation change significantly with gestation age and are influenced by the structural maturation of the sarcomere and changes in contractile filament protein isoforms., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

About two-thirds of human congenital heart disease involves second heart field-derived structures. Histone-modifying enzymes, histone deacetylases (HDACs), regulate the epigenome; however, their functions within the second heart field remain elusive. Here we demonstrate that histone deacetylase 3 (HDAC3) orchestrates epigenetic silencing of Tgf-β1, a causative factor in congenital heart disease pathogenesis, in a deacetylase-independent manner to regulate development of second heart field-derived structures. In murine embryos lacking HDAC3 in the second heart field, increased TGF-β1 bioavailability is associated with ascending aortic dilatation, outflow tract malrotation, overriding aorta, double outlet right ventricle, aberrant semilunar valve development, bicuspid aortic valve, ventricular septal defects, and embryonic lethality. Activation of TGF-β signaling causes aberrant endothelial-to-mesenchymal transition and altered extracellular matrix homeostasis in HDAC3-null outflow tracts and semilunar valves, and pharmacological inhibition of TGF-β rescues these defects. HDAC3 recruits components of the PRC2 complex, methyltransferase EZH2, EED, and SUZ12, to the NCOR complex to enrich trimethylation of Lys-27 on histone H3 at the Tgf-β1 regulatory region and thereby maintains epigenetic silencing of Tgf-β1 specifically within the second heart field-derived mesenchyme. Wild-type HDAC3 or catalytically inactive HDAC3 expression rescues aberrant endothelial-to-mesenchymal transition and epigenetic silencing of Tgf-β1 in HDAC3-null outflow tracts and semilunar valves. These findings reveal that epigenetic dysregulation within the second heart field is a predisposing factor for congenital heart disease., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Proper blood flow patterns are critical for normal cardiac morphogenesis, a process that occurs rapidly in order to support further development of all tissue and organs. Previously, intracardiac fluid forces have been shown to play a critical role in cardiac morphogenesis. Altered blood flow in early development can result in an array of cardiac defects including ventricular septal defects, valve malformations, and impaired cardiac looping. However, given the dynamic and highly transient nature of cardiac morphogenesis, time dependency of the mechanical environment as an epigenetic factor in relation to intracardiac forces must be significant. Here, we show that abnormal cardiac loading adversely influences cardiac morphology only during certain time windows, thus confirming that mechanical factors are a time-dependent epigenetic factor. To illustrate this, groups of zebrafish embryos were spaced at 6-h increments from 24 to 48 h post-fertilization (hpf) in which embryos were centrifuged to generate a noninvasive alteration of cardiac preload in addition to an overall hypergravity environment. We found that earlier and later treatment groups responded with altered morphology and function, while the group with altered preload from 30 to 36 hpf had no effect. These results demonstrate the inherently time-dependent nature of epigenetic factors as pertaining to intracardiac forces and external mechanical factors. Further, it underscores the highly coupled nature of programmed biology and mechanical forces during cardiac morphogenesis. Future studies with respect to surgical correction during cardiac morphogenesis must consider timing to optimize therapeutic impact.

The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A mathematical analysis of correlation of slow-wave processes in the system <> during the last 4 days of bearing was performed in experiments on rat fetuses with retained placental connection with the female. The parallel recording of physiological indicators of the female and fetus state revealed the existence of a relationship between oscillations of heartbeat rhythms and breathing in about-one-minute and many-minute ranges. The highest values of connection between the heart rhythms of female and fetus are characteristic for days 17 and 20 of gestation. On day 18-19 the interrelationships are slightly weaker. The specific mechanism providing this synchronization between heartbeat oscillations of mother and fetus is unclear. There are two hypothetic possibilities: an oscillation driver close in parameters for mother and fetus, and the maternal rhythm directly affecting the fetus.

Objective: Our aim is to evaluate the feasibility to examine the morphology and area of the atrioventricular (AV) valves in normal fetuses and fetuses with cardiac defects using spatiotemporal image correlation (STIC)., Methods: Atrioventricular valves were analyzed longitudinally in STIC volumes of 74 normal fetuses between the 15th and 36th week of pregnancy. The valve area was measured in a rendered view in diastole, the number of valve leaflets in systole. Longitudinal data analysis was performed using linear mixed models. Fifty fetuses with cardiac defects were examined., Results: Examination of 355 STIC volumes of normal fetuses showed in 82.5% sufficient quality. The tricuspid valve leaflets were seen in 200 (68.3%) volumes and the mitral valve leaflets in 219 (74.7%) volumes. The tricuspid valve showed in 61.1% a round, 29.0% rectangle, and 8.9% elliptical shape and the mitral valve in 60.1% round, 28.0% rectangle, and 10.9% elliptical. Regression analysis revealed a positive relationship of the valve area with gestational age (p < 0.0001). Most heart defects with stenosis showed an area below the 5th percentile., Conclusion: Prenatal examination of the morphology and area of the AV valves using four-dimensional ultrasound is feasible. A rectangular valve opening is normal, which was visualized in about one third of the normal fetuses., (© 2015 John Wiley & Sons, Ltd.)

Congenital heart disease (CHD) is among the most common congenital abnormalities. The prevalence of CHD ranges between 0.6% and 1.2% of live births. Despite its high prevalence, CHD is also among the most commonly missed abnormalities during prenatal ultrasound examination. A simple yet systematic approach to fetal heart examination, regular feedback, and implementation of training programs could improve detection rates and, in turn, neonatal outcome., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objectives: We aimed to construct reference ranges for time intervals of each component of cardiac flow velocity waveforms in normal fetuses, comparing those variables between right and left ventricles., Methods: In 359 fetuses at the gestational age of 17-38 weeks, the durations of atrioventricular (AV) valve opening (AVVO), AV valve closure (AVVC), total E- (total-E) and A- (total-A) waves, total ejection time (total-ET), acceleration time (acc-E for E-wave, acc-A for A-wave, and acc-ET for ejection time), and deceleration time (dec-E for E-wave, dec-A for A-wave, and dec-ET for ejection time) were studied cross sectionally., Results: Both right and left acc-E showed the strongest correlations with gestational age (r = 0.478 and r = 0.519, respectively). Left AVVO showed a stronger correlation (r = 0.474) than right AVVO (r = 0.282) and, conversely, right AVVC showed a stronger correlation (r = 0.399) than left AVVC (r = 0.195) with gestational age. Significant differences (all P values <0.001) were observed for all right and left parameters other than total-A and acc-E., Conclusions: Characteristic differences between right and left ventricles were found in the reference ranges, suggesting the developmental properties of the fetal heart. © 2014 John Wiley & Sons, Ltd., (© 2014 John Wiley & Sons, Ltd.)

In the developing heart, cardiomyocytes undergo terminal differentiation during a critical window around birth. Hypoxia is a major stress to preterm infants, yet its effect on the development and maturation of the heart remains unknown. We tested the hypothesis in a rat model that newborn anoxia accelerates cardiomyocyte terminal differentiation and results in reduced cardiomyocyte endowment in the developing heart via an endothelin-1-dependent mechanism. Newborn rats were exposed to anoxia twice daily from postnatal day 1 to 3, and hearts were isolated and studied at postnatal day 4 (P4), 7 (P7), and 14 (P14). Anoxia significantly increased HIF-1α protein expression and pre-proET-1 mRNA abundance in P4 neonatal hearts. Cardiomyocyte proliferation was significantly decreased by anoxia in P4 and P7, resulting in a significant reduction of cardiomyocyte number per heart weight in the P14 neonates. Furthermore, the expression of cyclin D2 was significantly decreased due to anoxia, while p27 expression was increased. Anoxia has no significant effect on cardiomyocyte binucleation or myocyte size. Consistently, prenatal hypoxia significantly decreased cardiomyocyte proliferation but had no effect on binucleation in the fetal heart. Newborn administration of PD156707, an ETA-receptor antagonist, significantly increased cardiomyocyte proliferation at P4 and cell size at P7, resulting in an increase in the heart to body weight ratio in P7 neonates. In addition, PD156707 abrogated the anoxia-mediated effects. The results suggest that hypoxia and anoxia via activation of endothelin-1 at the critical window of heart development inhibits cardiomyocyte proliferation and decreases myocyte endowment in the developing heart, which may negatively impact cardiac function later in life.

Background: Mouse mutants are used to model human congenital cardiovascular disease. Few studies exist comparing normal cardiovascular development in mice vs. humans. We carried out a systematic comparative analysis of mouse and human fetal cardiovascular development., Methods: Episcopic fluorescence image capture (EFIC) was performed on 66 wild-type mouse embryos from embryonic day (E) 9.5 to birth; 2-dimensional and 3-dimensional datasets were compared with EFIC and magnetic resonance images from a study of 52 human fetuses (Carnegie stage 13-23)., Results: Time course of atrial, ventricular, and outflow septation were outlined and followed a similar sequence in both species. Bilateral venae cavae and prominent atrial appendages were seen in the mouse fetus; in human fetuses, atrial appendages were small, and a single right superior vena cava was present. In contrast to humans with separate pulmonary vein orifices, a pulmonary venous confluence with one orifice enters the left atrium in mice., Conclusion: The cardiac developmental sequences observed in mouse and human fetuses are comparable, with minor differences in atrial and venous morphology. These comparisons of mouse and human cardiac development strongly support that mouse morphogenesis is a good model for human development.

Objective: The goal of our study was to evaluate the evolution of cardiac findings and assess the pregnancy outcome of fetuses with cardiac asymmetry at midgestation., Methods: We reviewed all fetuses with cardiac asymmetry at echocardiograms performed at 18-22 weeks of gestation from 2006 to 2013. The data collected included the gestational age at diagnosis, serial echocardiographic findings, karyotype testing, and pregnancy outcome. Excluded were cases of classical hypoplastic left heart syndrome (HLHS) at initial echocardiogram (i.e., aortic (AO) and/or mitral atresia or dysplasia with normal intracardiac connections), heterotaxy syndrome, twin pregnancy, fetal growth restriction, and coarctation of the aorta., Results: Our study included 278 fetuses with cardiac asymmetry (defined as Z-scores of left ventricle length or diameter of less than -2). A total of 202 (72%) fetuses had normal variations in cardiac growth by prenatal and neonatal echocardiography, 69 (25%) fetuses were confirmed to have HLHS variant by autopsy or neonatal echocardiography (with an overall survival of 4.3%), and seven (3%) fetuses were terminated before 24 weeks of gestation because of abnormal karyotypes. None of the cardiac measurements at the initial echocardiogram was significantly different between HLHS variant and normal cases. In the 69 fetuses ultimately diagnosed with HLHS variant, but not in the 202 normals, the Z-scores of all measured left heart structures decreased progressively between the first and the last prenatal echocardiograms (P < 0.01)., Conclusions: The majority of the fetuses with cardiac asymmetry at 18-22 weeks have a good outcome. A minority of them progress to develop HLHS variant with advancing gestation., (© 2014 John Wiley & Sons, Ltd.)

The primitive cardiac tube starts beating 6-8 weeks post fertilization in the developing embryo. In order to describe normal cardiac development during late first and early second trimester in human fetuses this study used microarray and pathways analysis and created a corresponding 'normal' database. Fourteen fetal hearts from human fetuses between 10 and 18 weeks of gestational age (GA) were prospectively collected at the time of elective termination of pregnancy. RNA from recovered tissues was used for transcriptome analysis with Affymetrix 1.0 ST microarray chip. From the amassed data we investigated differences in cardiac development within the 10-18 GA period dividing the sample by GA in three groups: 10-12 (H1), 13-15 (H2) and 16-18 (H3) weeks. A fold change of 2 or above adjusted for a false discovery rate of 5% was used as initial cutoff to determine differential gene expression for individual genes. Test for enrichment to identify functional groups was carried out using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Array analysis correctly identified the cardiac specific genes, and transcripts reported to be differentially expressed were confirmed by qRT-PCR. Single transcript and Ontology analysis showed first trimester heart expression of myosin-related genes to be up-regulated >5-fold compared with second trimester heart. In contrast the second trimester hearts showed further gestation-related increases in many genes involved in energy production and cardiac remodeling. In conclusion, fetal heart development during the first trimester was dominated by heart-specific genes coding for myocardial development and differentiation. During the second trimester, transcripts related to energy generation and cardiomyocyte communication for contractile coordination/proliferation were more dominant. Transcripts related to fatty acid metabolism can be seen as early as 10 weeks and clearly increase as the heart matures. Retinol receptor and gamma-aminobutyric acid (GABA) receptor transcripts were detected, and have not been described previously in human fetal heart during this period. For the first time global gene expression of heart has been described in human samples to create a database of normal development to understand and compare with known abnormal fetal heart development., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Chloride Intracellular Channel 4 (CLIC4) is one of seven members in the closely related CLIC protein family. CLIC4 is involved in multiple cellular processes including apoptosis, cellular differentiation, inflammation and endothelial tubulogenesis. Despite over a decade of research, no comprehensive in situ expression analysis of CLIC4 in a living organism has been reported. In order to fulfill this goal, we generated a knock-in mouse to express Green Fluorescent Protein (GFP) from the CLIC4 locus, thus substituting the GFP coding region for CLIC4. We used GFP protein expression to eliminate cross reaction with other CLIC family members., Results: We analyzed CLIC4 expression during embryonic development and adult organs. During mid and late gestation, CLIC4 expression is modulated particularly in fetal brain, heart, thymus, liver and kidney as well as in developing brown adipose tissue and stratifying epidermis. In the adult mouse, CLIC4 is highly expressed globally in vascular endothelial cells as well as in liver, lung alveolar septae, pancreatic acini, spermatogonia, renal proximal tubules, cardiomyocytes and thymic epithelial cells. Neural expression included axonal tracks, olfactory bulb, Purkinje cell layer and dentate gyrus. Renal CLIC4 expression was most pronounced in proximal tubules, although altered renal function was not detected in the absence of CLIC4. Myeloid cells and B cells of the spleen are rich in CLIC4 expression as are CD4 and CD8 positive T cells., Conclusions: In a comprehensive study detailing CLIC4 expression in situ in a mouse model that excludes cross reaction with other family members, we were able to document previously unreported expression for CLIC4 in developing fetus, particularly the brain. In addition, compartmentalized expression of CLIC4 in specific adult tissues and cells provides a focus to explore potential functions of this protein not addressed previously.

Barker's concept of 'foetal programming' proposes that intrauterine growth restriction (IUGR) predicts complex metabolic diseases through relationships that may be further modified by the postnatal environment. Dietary restriction and deficit in methyl donors, folate, vitamin B12, and choline are used as experimental conditions of foetal programming as they lead to IUGR and decreased birth weight. Overfeeding and deficit in methyl donors increase central fat mass and lead to a dramatic increase of plasma free fatty acids (FFA) in offspring. Conversely, supplementing the mothers under protein restriction with folic acid reverses metabolic and epigenomic phenotypes of offspring. High-fat diet or methyl donor deficiency (MDD) during pregnancy and lactation produce liver steatosis and myocardium hypertrophy that result from increased import of FFA and impaired fatty acid β-oxidation, respectively. The underlying molecular mechanisms show dysregulations related with similar decreased expression and activity of sirtuin 1 (SIRT1) and hyperacetylation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). High-fat diet and overfeeding impair AMPK-dependent phosphorylation of PGC-1α, while MDD decreases PGC-1α methylation through decreased expression of PRMT1 and cellular level of S-adenosyl methionine. The visceral manifestations of metabolic syndrome are under the influence of endoplasmic reticulum (ER) stress in overnourished animal models. These mechanisms should also deserve attention in the foetal programming effects of MDD since vitamin B12 influences ER stress through impaired SIRT1 deacetylation of HSF1. Taken together, similarities and synergies of high-fat diet and MDD suggest, therefore, considering their consecutive or contemporary influence in the mechanisms of complex metabolic diseases.