Feto-placental and coronary endothelial genes implicated in miscarriage, congenital heart disease and stillbirth, a systematic review and meta-analysis.

The first trimester placenta is very rarely investigated for placental vascular formation in developmental or diseased contexts. Defects in placental formation can cause heart defects in the fetus, and vice versa. Determining the causality is therefore difficult as both organs develop concurrently and express many of the same genes. Here, we performed a systematic review to determine feto-placental and coronary endothelial genes implicated in miscarriages, stillbirth and congenital heart defects (CHD) from human genome wide screening studies. 4 single cell RNAseq datasets from human first/early second trimester cardiac and placental samples were queried to generate a list of 1187 endothelial genes. This broad list was cross-referenced with genes implicated in the pregnancy disorders above. 39 papers reported feto-placental and cardiac coronary endothelial genes, totalling 612 variants. Vascular gene variants were attributed to the incidence of miscarriage (8 %), CHD (4 %) and stillbirth (3 %). The most common genes for CHD (NOTCH, DST, FBN1, JAG1, CHD4), miscarriage (COL1A1, HERC1), and stillbirth (AKAP9, MYLK), were involved in blood vessel and cardiac valve formation, with roles in endothelial differentiation, angiogenesis, extracellular matrix signaling, growth factor binding and cell adhesion. NOTCH1, AKAP12, CHD4, LAMC1 and SOS1 showed greater relative risk ratios with CHD. Many of the vascular genes identified were expressed highly in both placental and heart EC populations. Both feto-placental and cardiac vascular genes are likely to result in poor endothelial cell development and function during human pregnancy that leads to higher risk of miscarriage, congenital heart disease and stillbirth.

Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2.

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hypertrophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocardiography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Resolution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell-cycle defects and mitochondrial dysfunction. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteasomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteasomal system is activated; a mechanism that has been implicated in cardiomyopathies previously. In parallel, a lack of functional alpha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell-cycle defects, the likely cause of the death of the embryos. The defects also have wide-ranging morphological consequences.

Maternal choline supplementation in a rat model of periconceptional alcohol exposure: Impacts on the fetus and placenta.

BACKGROUND: Maternal choline supplementation in rats can ameliorate specific neurological and behavioral abnormalities caused by alcohol exposure during pregnancy. We tested whether choline supplementation ameliorates fetal growth restriction and molecular changes in the placenta associated with periconceptional ethanol exposure (PCE) in the rat. METHODS: Sprague Dawley dams were given either 12.5% ethanol (PCE) or 0% ethanol (Con) in a liquid diet from 4 days prior to 4 days after conception. At day 5 of pregnancy, dams were either placed on a standard chow (1.6 g choline/kg chow) or an intermediate chow (2.6 g choline/kg chow). On day 10 of pregnancy, a subset of the intermediate dams were placed on a chow further supplemented with choline (7.2 g choline/kg chow), resulting in 6 groups. Fetuses and placentas were collected on day 20 of pregnancy for analysis. RESULTS: Choline supplementation resulted in increased fetal weight at late gestation, ameliorating the deficits due to PCE. This was most pronounced in litters on a standard chow during pregnancy. Choline also increased fetal liver weight and decreased fetal brain:liver ratio, independent of alcohol exposure. Placental weight was reduced as choline levels in the chow increased, particularly in female placentas. This resulted in a greater ratio of fetal:placental weight, suggesting increased placental efficiency. Global DNA methylation in the placenta was altered in a sex-specific manner by both PCE and choline. However, the increased glycogen deposition in female placentas, previously reported in this PCE model, was not prevented by choline supplementation. CONCLUSIONS: Our results suggest that choline has the potential to ameliorate fetal growth restriction associated with PCE and improve placental efficiency following prenatal alcohol exposure. Our study highlights the importance of maternal nutrition in moderating the severity of adverse fetal and placental outcomes that may arise from prenatal alcohol exposure around the time of conception.

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.

Analysis of Placental Arteriovenous Formation Reveals New Insights Into Embryos With Congenital Heart Defects.

The placental vasculature provides the developing embryo with a circulation to deliver nutrients and dispose of waste products. However, in the mouse, the vascular components of the chorio-allantoic placenta have been largely unexplored due to a lack of well-validated molecular markers. This is required to study how these blood vessels form in development and how they are impacted by embryonic or maternal defects. Here, we employed marker analysis to characterize the arterial/arteriole and venous/venule endothelial cells (ECs) during normal mouse placental development. We reveal that placental ECs are potentially unique compared with their embryonic counterparts. We assessed embryonic markers of arterial ECs, venous ECs, and their capillary counterparts-arteriole and venule ECs. Major findings were that the arterial tree exclusively expressed Dll4, and venous vascular tree could be distinguished from the arterial tree by Endomucin (EMCN) expression levels. The relationship between the placenta and developing heart is particularly interesting. These two organs form at the same stages of embryogenesis and are well known to affect each other's growth trajectories. However, although there are many mouse models of heart defects, these are not routinely assessed for placental defects. Using these new placental vascular markers, we reveal that mouse embryos from one model of heart defects, caused by maternal iron deficiency, also have defects in the formation of the placental arterial, but not the venous, vascular tree. Defects to the embryonic cardiovascular system can therefore have a significant impact on blood flow delivery and expansion of the placental arterial tree.

Periconceptional ethanol exposure induces a sex specific diuresis and increase in AQP2 and AVPR2 in the kidneys of aged rat offspring.

Maternal alcohol consumption can impair renal development and program kidney dysfunction in offspring. Given that most women who drink alcohol cease consumption upon pregnancy recognition, we aimed to investigate the effect of alcohol around the time of conception (PC:EtOH) on offspring renal development and function. Rats received a liquid diet ±12.5% v/v ethanol from 4 days before to 4 days after mating. At postnatal day 30, nephron number was assessed. Urine flow and electrolyte (Na, K, Cl) excretion was measured at 6 and 19 months and blood pressure at 12 months. At 19 months, kidneys were collected for gene and protein analysis and assessment of collecting duct length. At postnatal day 30, PC:EtOH offspring had fewer nephrons. At 6 months, PC:EtOH exposure did not alter urine flow nor affect blood pressure at 12 months. At 19 months, female but not male offspring exposed to PC:EtOH drank more water and had a higher urine flow despite no differences in plasma arginine vasopressin (AVP) concentrations. Aqp2 mRNA and Avpr2 mRNA and protein expression was increased in kidneys from female PC:EtOH offspring but collecting duct lengths were similar. Immunofluorescent staining revealed diffuse cytoplasmic distribution of AQP2 protein in kidneys from PC:EtOH females, compared with controls with apical AQP2 localization. PC:EtOH resulted in a low nephron endowment and in female offspring, associated with age-related diuresis. Changes in expression and cellular localization of AQP2 likely underpin this disturbance in water homeostasis and highlight the need for alcohol to be avoided in early pregnancy.

Periconceptional alcohol exposure causes female-specific perturbations to trophoblast differentiation and placental formation in the rat.

The development of pathologies during pregnancy, including pre-eclampsia, hypertension and fetal growth restriction (FGR), often originates from poor functioning of the placenta. In vivo models of maternal stressors, such as nutrient deficiency, and placental insufficiency often focus on inadequate growth of the fetus and placenta in late gestation. These studies rarely investigate the origins of poor placental formation in early gestation, including those affecting the pre-implantation embryo and/or the uterine environment. The current study characterises the impact on blastocyst, uterine and placental outcomes in a rat model of periconceptional alcohol exposure, in which 12.5% ethanol is administered in a liquid diet from 4 days before until 4 days after conception. We show female-specific effects on trophoblast differentiation, embryo-uterine communication, and formation of the placental vasculature, resulting in markedly reduced placental volume at embryonic day 15. Both sexes exhibited reduced trophectoderm pluripotency and global hypermethylation, suggestive of inappropriate epigenetic reprogramming. Furthermore, evidence of reduced placental nutrient exchange and reduced pre-implantation maternal plasma choline levels offers significant mechanistic insight into the origins of FGR in this model.

Effects of periconceptional maternal alcohol intake and a postnatal high-fat diet on obesity and liver disease in male and female rat offspring.

The effects of maternal alcohol consumption around the time of conception on offspring are largely unknown and difficult to determine in a human population. This study utilized a rodent model to examine if periconceptional alcohol (PC:EtOH) consumption, alone or in combination with a postnatal high-fat diet (HFD), resulted in obesity and liver dysfunction. Sprague-Dawley rats were fed a control or an ethanol-containing [12.5% (vol/vol) EtOH] liquid diet from 4 days before mating until 4 days of gestation ( n = 12/group). A subset of offspring was fed a HFD between 3 and 8 mo of age. In males, PC:EtOH and HFD increased total body fat mass ( PPC:EtOH < 0.05, PHFD < 0.0001); in females, only HFD increased fat mass ( PHFD < 0.0001). PC:EtOH increased microvesicular liver steatosis in male, but not female, offspring. Plasma triglycerides, HDL, and cholesterol were increased in PC:EtOH-exposed males ( PPC:EtOH < 0.05), and LDL, cholesterol, and leptin (Lep) were increased in PC:EtOH-exposed females ( PPC:EtOH < 0.05). mRNA levels of Tnf-α and Lep in visceral adipose tissue were increased by PC:EtOH in both sexes ( PPC:EtOH < 0.05), and Il-6 mRNA was increased in males ( PPC:EtOH < 0.05). These findings were associated with reduced expression of microRNA-26a, a known regulator of IL-6 and TNF-α. Alcohol exposure around conception increases obesity risk, alters plasma lipid and leptin profiles, and induces liver steatosis in a sex-specific manner. These programmed phenotypes were similar to those caused by a postnatal HFD, particularly in male offspring. These results have implications for the health of offspring whose mothers consumed alcohol around the time of conception.