Weeks Of Human Gestation Research Articles

Normal testicular function and spermatogenesis

The testis performs two basic functions, sperm production and testosterone secretion. Formation of the testis is genetically controlled; expression of the SRY gene directs the embryonic gonads into the pathway leading to the development of testes. By the fourth week of gestation in humans, the primordial germ cells derived from pluripotent cells of the embryonic epiblast proliferate and migrate from the endoderm of the yolk sac into the undifferentiated gonad, which becomes morphologically distinct during the seventh week of gestation in humans. Histological development of the testis is largely completed by the end of the third month of gestation.

Unraveling the Genetic Landscape of Bladder Development in Mice

To better understand the pathobiology of human congenital bladder abnormalities and disorders associated with dedifferentiation, such as bladder cancer, we must first unravel the biology of normal bladder development. Therefore, we performed microarray analysis focusing on determining the gene expression profile at the initiation of bladder development. RNA was extracted from embryonic day 13 and 18 mouse bladders (anatomically equivalent to 7 and 13 weeks of human gestation) and gene expression was evaluated using microarrays. Alterations in select genes of biological interest were confirmed using real-time quantitative polymerase chain reaction and localization was determined by immunohistochemistry. The genetic profile in the initiating mouse bladder at embryonic day 13 was dominated by transcription factors, retinoic acid signaling genes, Eph/ephrin bidirectional signaling molecules and genes associated with regulating cell cycle and differentiation. Later in development at embryonic day 18 genes associated with smooth muscle, innervation and epithelial differentiation were up-regulated. In addition, we examined the functional role of midkine, which was highly expressed at embryonic day 13, using organ culture and to our knowledge we provide the first evidence that this growth factor up-regulates molecules associated with bladder smooth muscle differentiation. These data provide novel insights into molecules that orchestrate bladder development and highlight genes that may be involved in diseases associated with abnormal differentiation.

A possible explanation of what's happening at the moment of palatal shelf elevation

Cleft lip and palate is multifactorial in aetiology. The elevation of palatal shelves is a key point of palatogenesis. However, there were many different opinions on the explanation of the elevation. In this article, we offered a new explanation. Before sixth week of gestation in humans, Palatal mesenchymal proliferation was along the horizontal direction. Because of the block of the tongue, the palatal shelves had to grow first vertically in the oral cavity. In the process of cells migration, much horizontal stress accumulated in the palatal shelves, meanwhile increased the collagen secretion of the palatal mesenchymal cells in order to strengthen the elasticity of palatal shelf and maintain the integrity to make palatal shelf look like an elastic palate. The intrinsic elevating force and the block of tongue made the palatal shelf curved. After seventh week facial structures grew predominantly in the sagittal plane. The activity of the geniohyoid and genioglossus muscles caused the mandibular retraction and the widening of the angulation between the bilateral hemimandibles. These changes provided the space for palatal shelf elevation. At some moment of the eighth to tenth weeks, the elastic stress center of the palatal shelf was above the horizontal surface because of the drop of the tongue. The palatal shelves might bounce up and elevate in a horizontal position when enough horizontal stress accumulated, and then adhered and fused.

SCHU-29: Notch-3 and Notch-4 in the Development of a Normal Prostate Gland in a Mouse

The Notch signalling is a well-conserved system that has been found from invertebrates to mammals including humans. Notch signalling is found on the cell surface and when a specific ligand on the surface of the neighbours cells are expressed, Notch activates a complex pathway which results in the transcription of specific DNA sequences. This determines the cell fate, and also affects proliferation, differentiation, apoptosis and inter-cellular adhesion. During early developmental stages of the normal prostate, (approximately 10 weeks of gestation in humans), outbreaks of epithelium invade the mesenchyma, generating a mutual induction of differentiation.

Lack of effect of antenatal glucocorticoid therapy in the fetal baboon on cerebral cortical glucose transporter proteins

Maternal antenatal glucocorticoid therapy is used to accelerate lung maturation of immature babies at risk of preterm delivery. It acutely affects brain activity of the human fetus and reduces the immunoreactivity of neurocytoskeletal and synaptic proteins in the fetal baboon brain. These effects might be based on cerebral energy failure due to a decreased neuronal glucose uptake that has been shown in vitro. Glucose uptake into the brain is selectively facilitated by GLUT1 expressed in the blood-brain barrier and GLUT3 expressed in the neuronal membrane. Immunohistochemical distribution of GLUT1 and GLUT3 were examined in the frontal neocortex of the fetal baboon brain at 0.73 gestation (i.e. similar to 28 weeks of human gestation) after maternal betamethasone administration, mimicking the clinical dose regimen. Betamethasone did not alter GLUT1 and GLUT3 immunoreactivity. The results suggest that inhibition of glucose uptake is not the mechanism for the cerebral effects of antenatal glucocorticoids.

Factors affecting the early embryonic environment

The early human embryo develops in a tightly controlled, relatively protected environment. During the first 8–12 weeks of human gestation, the delivery of maternal blood, and therefore the amount of oxygen to the developing embryo is limited and the embryo is supplied with essential nutrients in part via the decidual uterine glands, the placental trophoblast and the secondary yolk sac. Factors that interfere with this process may well result in spontaneous miscarriage or adverse outcome later in pregnancy. There is mounting evidence for the presence of transporter systems for many substances including drugs and toxins from the maternal to the fetal compartments in early pregnancy. The role of these substances in both the protection of early pregnancy development and possible teratogenicity are explored in this chapter. Clearly, the timing of exposure to potentially damaging substances is crucial with effects on conception, implantation, placental development and organogenesis dependent on the gestation at which exposure occurs.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

Investigation of Cerebral Development and Injury in the Prematurely Born Primate by Magnetic Resonance Imaging and Histopathology

We summarize the preliminary results of brain histopathology and magnetic resonance imaging applied to a premature baboon model developed for evaluation of the pathogenesis and treatment of bronchopulmonary dysplasia. Cerebral development was assessed in 10 gestational control animals at time points of 125, 140 and 160 days of gestation (dg). On the basis of histopathology, conventional MRI and diffusion MRI, 125 dg is equivalent to 26–28 weeks of human gestation, 140 dg is equivalent to 30–32 weeks, and 160 dg is equivalent to term. Preliminary data are also presented for 33 experimental cases delivered at 125 dg, nursed for 2 weeks in an intensive care facility, and sacrificed at 139–140 dg. The commonest neuropathology in this cohort is white matter damage, manifest by reactive astrogliosis or activated microglia, and enlarged ventricular size. Subarachnoid, germinal matrix and intraventricular hemorrhages are also common. These preliminary results support the similarity of this model to the human preterm infant for both cerebral development and the pattern of cerebral injury. The prematurely born baboon appears an important model for the study of preterm human birth.

Hyperglycemia: its imminent effects on mammalian nephrogenesis

A sustained exposure of the mammalian embryo to very high glucose ambience is associated with a multitude of congenital birth defects, including those of the cardiovascular, CNS, skeletal and urogenital systems during the first 6-8 weeks of gestation in humans. These urogenital abnormalities may be associated with "caudal regression syndrome" or may occur alone in the form of partial or total renal agenesis. Similarly, an increase in the incidence of morphogenetic defects is observed in the offspring of streptozotocin-induced diabetic rats and mice, and also in non-obese diabetic mice. In certain cases, failure during the growth of the lower parts of embryos or newborn mice involving the genitourinary system has been observed in animals with severe diabetes. Investigators have utilized whole organ culture systems to delineate the mechanisms relevant to dysmorphogenesis of the embryonic metanephros. A marked dysmorphogenesis of the metanephros is observed upon treatment with a high concentration of D: -glucose. Associated with it are changes that include branching dysmorphogenesis of the ureteric bud iterations, reduced population of nascent nephrons, decreased expression of basement membrane proteoglycans, depletion of ATP stores, and fulminant apoptosis of the cells at the interface of mesenchyme and ureteric bud epithelium. The latter findings suggest that disruption of epithelial:mesenchymal interactions may be the major event responsible for the metanephric dysmorphogenesis induced by high glucose ambience.

Renal development in high-glucose ambience and diabetic embryopathy

Maternal diabetes has an adverse influence on the intrauterine growth of the fetus, which is attributable to the exposure of the mammalian embryo to an abnormal metabolic environment. A sustained exposure of the fetus to such an environment (ie, elevated concentration of glucose), during the first 6 to 8 weeks of gestation in humans may result in diabetic embryopathy, which is characterized by a multitude of congenital birth defects, including those of the nervous, cardiovascular, skeletal, and urogenital systems. The urogenital abnormalities may be associated with caudal regression syndrome or may occur alone in the form of partial or total renal agenesis. Similarly, an increase in the incidence of morphogenetic defects is observed in offsprings of streptozotocin-induced diabetic rats and mice and also in nonobese diabetic mice. In certain instances, failure in the growth of lower part of embryos or newborn mice has been observed in animals with a severe diabetic state. For further delineation of the mechanisms involved in the pathogenesis of diabetic embryopathy, the investigators used whole-embryo culture systems, and found that glucose can induce defects mainly confined to the lower part of the body involving the genitourinary system. Similarly, dysmorphogenesis of the embryonic metanephros is observed when it is subjected to high concentrations of D-glucose and its epimer D-mannose. This article discusses certain aspects of diabetic embryopathy with an emphasis on changes that occur in the fetal metanephros in high-glucose ambience.

Alterations in Human Placental 11β-hydroxysteroid Dehydrogenase Type 1 and 2 with Gestational Age and Labour

11β-hydroxysteroid dehydrogenase type 1 and type 2 may be important in the process of human parturition and the regulation of fetal growth, by the modulation of cortisol concentrations in the fetal compartment. Changes in the expression and activity of these enzymes in late gestation have not been well described. This study has examined the gene expression of placental 11β-HSD1 and 2, activity of 11β-HSD2 and fetal cortisol concentrations during the final few weeks of human pregnancy and with the onset of labour. Placental 11β-HSD2 activity decreased significantly between 38 and 40 weeks. There were no significant changes in mRNA abundance or protein expression with gestational age or labour. Placental 11β-HSD1 mRNA abundance significantly increased with spontaneous labour. Fetal cortisol concentrations increased significantly with spontaneous labour. This study is the first to describe a decrease in 11β-HSD2 activity in the last few weeks of human gestation. This decrease in type 2 activity, along with an increase in 11β-HSD1 gene expression may be a mechanism by which cortisol concentrations rise at term to regulate fetal maturation and activate pathways associated with labour.

Factors Influencing Fetal Growth

After completing this article, readers should be able to: 1. Describe the roles of insulin-like growth factor on fetal growth and development. 2. Delineate components of “maternal constraint.” 3. Explain the role of the placenta in regulating fetal growth. 4. Describe the effects of fetal glucose, amino acid, and fat supply on fetal growth. The principal determinants of fetal growth are fetal genotype and in utero environment. Environmental factors include maternal and paternal genetics, maternal size, and the capacity of the placenta to provide nutrients to the fetus. These environmental factors interact with the intrinsic growth pattern of the fetus, yielding a particular rate and composition of fetal growth. Most of the variation in fetal growth in a population is due to variations in environmental factors, not the fetal genome, although a genetically abnormal fetus clearly might not grow as well as a normal fetus if affected genes include those that are important for growth. Many genes contribute to fetal growth and birthweight. Techniques in which specific genes can either be deleted (“knockouts”) or overexpressed have led to greater understanding of how some of these genes regulate fetal growth. Such studies have shown that both maternal and paternal influences are present during fetal development and are passed on to the developing fetus by spermatozoa or oogonia by a mechanism called imprinting. Although the maternal genetic composition exerts greater influence than fetal genotype in the overall regulation of fetal growth, both maternal and paternal genomes are important in fetal growth and development (1)(2). For example, gynogenetic zygotes (two maternal genome copies) lead to underdeveloped extraembryonic tissues but well-developed embryos. The more modest regulation offered by the paternal genotype is essential for trophoblast development. For example, zygote nuclear transfer experiments have shown that androgenetic zygotes (two paternal genome copies) develop extensive trophoblast tissues but …

Gonadoblastoma, Testicular and Prostate Cancers, and the TSPY Gene

The role of the Y chromosome in oncogenesis of human cancers has been somewhat controversial. Both gain and loss of the Y chromosome in different leukemia, lymphoma, and solid tumors have been reported (Abeliovich et al. 1994; Brothman 1997; Dave et al. 1996; Geburek et al. 1997; Jackson-Cook et al. 1996; Kirk et al. 1994; Konig et al. 1994; Mertens et al. 1997; Riske et al. 1994; Watanabe et al. 1996). Conceivably, both oncogenes and tumor-suppressor genes exist on this chromosome, and they may act at different points during tumorigenesis, particularly for cancers of male-specific organs, such as the testis and the prostate.

Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo.

Hepatic hematopoiesis in the mouse embryo is preceded by two hematopoietic waves, one in the yolk sac, and the other in the paraaortic splanchnopleura, the presumptive aorta-gonad-mesonephros region that gives rise to prenatal and postnatal blood stem cells. An homologous intraembryonic site of stem cell emergence was previously identified at 5 weeks of human gestation, when hundreds of CD34(++ )Lin- high-proliferative potential hematopoietic cells border the aortic endothelium in the preumbilical region. In the present study, we have combined immunohistochemistry, semithin section histology, fluorescence-activated cell sorting and blood cell culture in an integrated study of incipient hematopoiesis in the human yolk sac, truncal arteries and embryonic liver from 21 to 58 days of development. The chronology of blood precursor cell emergence in these distinct tissues suggests a pivotal role in the settlement of liver hematopoiesis of endothelium-associated stem cell clusters, which emerge not only in the dorsal aorta but also in the vitelline artery. Anatomic features and in vitro functionality indicate that stem cells develop intrinsically to embryonic artery walls from a presumptive territory whose blood-forming potential exists from at least 24 days of gestation.

DEVELOPMENTAL EXPRESSION OF JAGGED1 IN THE MAMMALIAN HEART: IMPLICATIONS FOR CARDIAC DISEASE IN ALAGILLE SYNDROME

24 Alagille syndrome (AGS) is an autosomal dominant disorder characterized by developmental abnormalities of the liver, heart, eye, skeleton and kidney. Congenital heart defects, the majority of which are right-sided, contribute significantly to mortality in AGS patients. Mutations in Jagged1, a conserved gene in the Notch intercellular signaling pathway, have been found to cause AGS. In order to understand the role of Jagged1 in normal heart development and in the heart defects seen in AGS, we have studied the expression pattern of Jagged1 in the developing mammalian heart. Jagged1 expression was assayed by whole mount and section in situ hybridizations on mouse and human embryos. Whole mount in situ hybridizations of mouse embryos at 8.0 and 8.5 days post coitum (dpc), corresponding to 3 weeks of human gestation, reveal Jagged1 expression in the first pharyngeal arch. Jagged1 is expressed in the human heart at 8 weeks of gestation transmurally in the atria and in the endocardium of the ventricles. Expression is also detected in the epicardium, which is derived from liver primordium. In order to identify vascular structures in the mouse embryo at 10.5 dpc, in situ hybridization was performed with a probe for PECAM, an endothelial cell marker. Combined studies with probes for Jagged1 and PECAM show overlapping patterns of expression in the branchial arch arteries and descending aorta, confirming Jagged1 expression in these vascular structures. In situ hybridization of mouse embryos at 12.5 dpc shows Jagged1 expression in the endothelial cells of the endocardial cushions which will undergo mesenchymal transformation to form value tissue. Expression is particularly accentuated in the right-sided outflow tract, ductus arteriosus, pulmonary arteries and lung mesoderm. We conclude that Jagged1 is expressed in the developing mammalian heart in multiple vascular structures and in the developing valves. This pattern of expression provides evidence that Jagged1 has a direct role in cardiac development and that the sites of expression correlate with the congenital heart defects seen in AGS.

Development of hepatic sinusoidal structure with special reference to the Ito cells.

To elucidate sinusoidal cell structure and function under normal conditions and their behavior in diseased settings, an understanding of their developmental aspects is needed. At day 10 of gestation in mice and rats or at 5 weeks of gestation in humans, the hepatic cords grow into the mesenchymal tissue of the septum transversum, and the primitive sinusoidlike structure is simultaneously observed between the liver cell cords. In the margin of the growing liver primordium, mesenchymal cells in the septum transversum are trapped in the subendothelial space. These subendothelial cells are at the early stages of organogenesis and become progenitors of the Ito cells. By days 12-14 of gestation in mice and rats or 8 weeks of gestation in humans, the basic structure of the sinusoids has developed. Embryonic hepatic sinusoids are usually lined by a continuous endothelium without basement membranes, and an incompletely fenestrated sinusoid appears at the middle gestational stage. In the late gestational stages, the Ito cells exhibit myofibroblastlike features in humans, mice, and rats. In association with this event, perisinusoidal reticular networks are gradually intensified. After birth until days 4-5 in mice and rats, the sinusoidal and perisinusoidal structures are almost completely formed, although slight morphological differences from those in adult livers still exist. What happens to sinusoidal endothelial cells and Ito cells in hepatic fibrosis-cirrhosis of the adult may be a deviated or uncontrolled occurrence of what goes on during the fetal period, i.e., a continuous nonfenestrated sinusoidal lining in the early embryonic stage and a myofibroblastlike transformation of Ito cells in late fetal life.

Reciprocal entorhinal-hippocampal connections established by human fetal midgestation.

Little is known about the timing or sequence of genesis of connections between different areas of the developing human cerebral cortex. It has been shown that connections between areas V1 and V2 of the visual isocortex are established at about 37 weeks of gestation (Burkhalter [1993] Cerebr. Cortex 3:476-487), suggesting that cortico-cortical connections appear late in the 40-week human gestational period. However, there are indications from other studies that connections between subdivisions of the hippocampal formation may be established much earlier, by about 20 weeks of human gestation. To investigate this possibility, the lipophilic bidirectional tracer 1,1' dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (DiI) was used to study connections between the entorhinal cortex, hippocampus, and temporal lobe neocortex in paraformaldehyde-fixed postmortem fetal tissue. The DiI transport revealed robust reciprocal connections between the entorhinal cortex, hippocampus, and subiculum, which were consistently present at 19 weeks of gestation (the earliest age studied), and which were anatomically similar to those in adult primates. Specifically, projections to the hippocampus and subiculum originated from neurons in the entorhinal cortex (EC) layers 2 and 3, whereas reciprocal projections to the EC originated from pyramidal neurons in the cornu ammonis region CA1 and the subiculum. In contrast, the perforant pathway projection from EC to the dentate gyrus, and all connections with the neocortex, reached only rudimentary stages of development by 22 weeks of gestation (the latest age studied). These findings suggest that hippocampal pathways develop prior to isocortical pathways, and that reciprocal entorhinal-hippocampal projections may be among the first cortico-cortical connections to be established in the human brain.

Stage-dependent effects of ethanol on cranial neural crest cell development: partial basis for the phenotypic variations observed in fetal alcohol syndrome.

Fetal alcohol syndrome (FAS) is characterized by growth retardation, mental deficiencies, and numerous craniofacial and neuronal anomalies; the type and severity of these defects may be related to the time and dose of maternal ethanol exposure. Ethanol administered during presomitic stages results in the typical FAS craniofacial phenotype and is accompanied by a loss of cranial neural crest cells (CNCCs) through ethanol-induced cell death. However, the stage-specific effects of ethanol on the CNCC population is unknown. We examined the effects of ethanol on CNCC populations by treating in ovo chick embryos with a single ethanol dose (0.43 mmol/egg) at various stages of CNCC development, and corresponding to the first 3-4 weeks of human gestation. Ethanol treatment induced cell death and reduced CNCC populations in patterns consistent with observed dysmorphologies of CNCC-derived cranial structures. The precise population affected was dependent on the timing of ethanol exposure. Treatment at gastrulation or neurulation induced cell death and losses of CNCC populations, particularly those in rostral positions, and resulted in more severe craniofacial defects. In contrast, treatment at early somitic stages (4-16 somites) induced cell death, primarily within caudal CNCC populations, but resulted in less severe craniofacial defects, suggesting an increased capacity for recovery. These results suggest that there are distinct developmental windows during which the CNCCs may be particularly susceptible to ethanol-induced cell death. We conclude that ethanol exposure seems to affect specific events adversely during neural crest development. The timing of embryonic ethanol exposure relative to CNCC development could account, in part, for the heterogenous craniofacial defects observed in FAS.

Osmoregulation of thirst and vasopressin release in pregnancy.

Osmoregulation is altered in human gestation, body tonicity declining to a nadir early in pregnancy after which a new steady-state plasma osmolality is maintained until term. Development of precise, sensitive, and specific radioimmunoassays for arginine vasopressin (AVP), which permit clearer definitions of functional properties of the osmoregulatory system, have led to a formulation of how these changes occur (both in women as well as in a gravid rat model). The osmotic thresholds for thirst and antidiuretic hormone release each decrease approximately 10 mosmol/kg during the initial weeks of human gestation. Lowering the threshold to drink stimulates increased water intake and dilution of body fluids. Because AVP release is not suppressed at the usual levels of tonicity, it still circulates and water is retained. Osmolality declines until it decreases below the osmotic thirst threshold (situated several mosmol/kg above that for hormone secretion), and a new steady state, with little change in water turnover, is established. The metabolic clearance rate of AVP is also altered, increasing three- to fourfold between gestational week 10 and midtrimester, paralleling the appearance and rapid rise in circulating cystine-aminopeptidase (vasopressinase), an observation that may explain several disorders of water handling that complicate human pregnancy. Finally, mechanisms responsible for the altered osmoregulation are obscure but chorionic gonadotropin may be involved in the changes during human gestation.

Fetal alcohol syndrome and DiGeorge anomaly: critical ethanol exposure periods for craniofacial malformations as illustrated in an animal model.

Acute maternal ethanl (alcohol) administration induces different craniofacial anomalies in the offspring of experimental animals, depending on the gestational day of teratogen exposure. Previous studies in our laboratories have illustrated the sequence of developmental changes leading to the "typical" fetal alcohol syndrome (FAS) craniofacial phenotype which results from teratogen exposure during gastrulation. These facial features are accompanied by deficiencies in median forebrain derivatives. Ethanol teratogenesis at this time apparently results in a loss of midline territory of the embryonic disc with little effects on neural crest-dependent laterally derived structures including the visceral arches. Acute ethanol exposure in mice 1 1/2 days later, at a time when neural crest cells are populating the frontonasal prominence and the visceral arches, results in a craniofacial phenotype that is similar to that noted in the DiGeorge anomaly or sequence. Sequential scanning electron microscopic analysis in our laboratory of embryos exposed on day 8 1/2 have illustrated deficiencies in the developing facial prominences and the visceral arches. The developing forebrain and midbrain appear hypoplastic. We have also observed heart, great vessel, and thymus abnormalities in these fetuses. Histologic analyses indicate that a common pathogenetic basis for the above-mentioned (day 8 1/2-induced) fetal alcohol effects appears to be an interference with the integrity of the cranial (including occipital) neural crest. Other discrete cell populations may also be involved since we have observed abnormalities in other regions, including placodal and closing membrane tissues. This animal model provides evidence linking maternal ethanol abuse during the 3rd or 4th weeks of human gestation to the development in the conceptus of FAS or DiGeorge anomally craniofacial characteristics, respectively. As the DiGeorge anomaly has been noted in the offspring of alcoholic women, this animal model indicates that ethanol and/or its metabolites is, in these cases, the causative agent.