Ryanodine receptor expression in trophoblasts

Trophoblasts of the placenta are the frontline cells involved in communication and exchange of materials between the mother and fetus. Within trophoblasts, calcium signalling proteins are richly expressed. Intracellular free calcium ions are a key second messenger, regulating various cellular activities. Transcellular Ca2+ transport through trophoblasts is essential in fetal skeleton formation. Ryanodine receptors (RyRs) are high conductance cation channels that mediate Ca2+ release from intracellular stores to the cytoplasm. To date, the roles of RyRs in trophoblasts have not been reported. By use of reverse transcription PCR and western blotting, the current study revealed that RyRs are expressed in model trophoblast cell lines (BeWo and JEG-3) and in human first trimester and term placental villi. Immunohistochemistry of human placental sections indicated that both syncytiotrophoblast and cytotrophoblast cell layers were positively stained by antibodies recognising RyRs; likewise, expression of RyR isoforms was also revealed in BeWo and JEG-3 cells by immunofluorescence microscopy. In addition, changes in [Ca2+]i were observed in both BeWo and JEG-3 cells upon application of various RyR agonists and antagonists, using fura-2 fluorescent videomicroscopy. Furthermore, endogenous placental peptide hormones, namely angiotensin II, arginine vasopressin and endothelin 1, were demonstrated to increase [Ca2+]i in BeWo cells, and such increases were suppressed by RyR antagonists and by blockers of the corresponding peptide hormone receptors. These findings indicate that 1) multiple RyR subtypes are expressed in human trophoblasts; 2) functional RyRs in BeWo and JEG-3 cells response to both RyR agonists and antagonists; 3) RyRs in BeWo cells mediate Ca2+ release from intracellular store in response to the indirect stimulation by endogenous peptides. These observations suggest that RyR contributes to trophoblastic cellular Ca2+ homeostasis; trophoblastic RyRs are also involved in the functional regulation of human placenta by coupling to endogenous placental peptide-induced signalling pathways.

Nutrition, growth and body composition in preterm infants

The goal of neonatal nutrition in the preterm infant is to achieve postnatal growth and body composition approximating that of a normal fetus of the same postmenstrual age and to obtain a functional outcome comparable to infants born at term. However, in clinical practice such a pattern is seldom achieved, with growth failure and altered body composition being extensively reported. The BabyGrow preterm nutrition study was a longitudinal, prospective, observational study designed to investigate nutrition and growth in 59 preterm infants following the implementation of evidence-based nutrition guidelines in the neonatal unit at Cork University Maternity Hospital. Nutrient delivery was precisely measured during the entire hospital stay and intakes were compared with current international recommendations. Barriers to nutrient delivery were identified across the phases of nutritional support i.e. exclusive parenteral nutrition and transition (establishment of enteral feeds) phases of nutrition and nutritional strategies to optimise nutrient delivery were proposed according to these phases. Growth was measured from birth up to 2 months corrected age and body composition was assessed in terms of fat mass and lean body mass by air displacement plethysmography (PEA POD) at 34 weeks gestation, term corrected age and 2 months corrected age. Anthropometric and body composition data in the preterm cohort were compared with a term reference group from the Cork BASELINE Birth Cohort Study (n=1070) at similar time intervals. The clinical and nutritional determinants of growth and body composition during the neonatal period were reported for the first time. These data have international relevance, informing authoritative agencies developing evidence-based practice guidelines for neonatal nutritional support. In the future, the nutritional management of preterm infants may need to be individualised to consider gestational age, birth weight as well as preterm morbidity.

Kinetics of anti-Campylobacter jejuni monomeric and polymeric immunoglobulin A1 and A2 responses in serum during acute enteritis.

The intensity and kinetics of the serum polymeric and monomeric immunoglobulin A1 (IgA1) and IgA2 antibody responses to Campylobacter jejuni were analyzed. A rapid and marked serum IgA antibody response involving both the monomeric and polymeric components of IgA was observed after C. jejuni infections. IgA antibodies reached a peak of activity in serum during week 2 after the first symptoms of enteritis, about 10 days before the peak of IgG activity. Polymeric IgA accounted for most of the anti-C. jejuni activity at the peak of the IgA response (median, 90%; range, 44 to 98%) but rapidly disappeared from serum over a few weeks. In contrast, the serum monomeric IgA antibody response was low and was maintained over a prolonged period of time. Anti-C. jejuni IgA detected in the serum of healthy blood donors was mainly monomeric (median, 83%; range, 17 to 94%). In both the patients and the positive controls, IgA1 was the predominant (greater than 85%) subclass involved, even when the IgA antibody response was mainly polymeric. Our results suggest that polymeric IgA antibody responses are linked to a strong or persisting antigenic stimulation or both. Polymeric IgA antibodies appear to be a potential marker of acute C. jejuni infections, and their determination could provide a useful tool for the serological diagnosis of recent C. jejuni infections.

Monitoring maternal Beta carotene and retinol consumption may decrease the incidence of neurodevelopmental disorders in offspring.

Retinoic acids (13-cis and 13-trans) are known teratogens, and their precursor is retinol, a form of vitamin A. In 1995, Rothman et al demonstrated an association between excessive vitamin A, >10,000 IU/day, during the first trimester of pregnancy and teratogenic effects, particularly in the central nervous system. However, vitamin A deficiency has long been known to be deleterious to the mother and fetus. Therefore, there may be a narrow therapeutic ratio for vitamin A during pregnancy that has not previously been fully appreciated. Neurodevelopmental disorders may not be apparent by macroscopic brain examination or imaging, and proving the existence of a behavioral teratogen is not straightforward. However, an excess of retinoic acid and some neurodevelopmental disorders are both associated with abnormalities in cerebellar morphology. Physical and chemical evidence strongly supports the notion that beta carotene crosses the placenta and is metabolized to retinol. Only very limited amounts of beta carotene are stored in fetal fat cells as evidenced by the fact that maternal fat is yellow from beta carotene, whereas non-brown neonatal fat is white. Furthermore, newborns of carotenemic mothers do not share the yellow complexion of their mothers. The excess 13-trans retinoic acid derived from metabolized beta carotene in the fetus increases the concentration of the more teratogenic 13-cis retinoic acid since the isomerization equilibrium is shifted to the left. Therefore, this paper proposes that consideration be given to monitoring all potential sources of fetal 13-cis and 13-trans retinoic acid, including nutritional supplements, dietary retinol, and beta carotene, particularly in the first trimester of pregnancy.

Le rôle de l'hormone anti-müillerienne dans la régulation du fonctionnement de l'ovaire. Revue de la littérature

Anti-mullerian hormone, also called AMH, belongs to the large family of transforming growth factor P. Its role in the sexual differentiation of male fetus is now well known. Recently, AMH has been demonstrated to play an important role in the ovarian function. In fact, AMH seems to regulate the kinetics of follicular development, inhibiting the follicular recruitment and the follicular growth. Thus, this intra-gonadic cybernin could be a decisive determinant of the rapidity of follicular pool exhaustion. Today, some experimental data from the literature suggest that AMH could be a reliable marker of ovarian reserve. This review summarizes the present knowledge about AMH and its role in physiology but also in ovarian pathology.

TRANSIENT LIPOPOLYSACCHARIDE-INDUCED CYTOKINE RESPONSES IN THE MATERNAL AND FETAL GUINEA PIG

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study.

Investigation of the effect of intrauterine inflammation and infection on fetal brain injury using human and animal models

In recent years, increased focus has been placed on the role of intrauterine infection and inflammation in the pathogenesis of fetal brain injury leading to neurodevelopmental disorders such as cerebral palsy. At present, the mechanisms by which inflammatory processes during pregnancy cause this effect on the fetus are poorly understood. Our previous work has indicated an association between experimentally-induced intrauterine infection, increased proinflammatory cytokines, and increased white matter injury in the guinea pig fetus. In order to further elucidate the pathways by which inflammation in the maternal system or the fetal membranes leads to fetal impairment, a number of studies investigating aspects of the disease process have been performed. These studies represent a body of work encompassing novel research and results in a number of human and animal studies. Using a guinea pig model of inflammation, increased amniotic fluid proinflammatory cytokines and fetal brain injury were found after a maternal inflammatory response was initiated using endotoxin. In order to more closely monitor the fetal response to chorioamnionitis, a model using the chronically catheterized fetal ovine was carried out. This study demonstrated the adverse effects on fetal white matter after intrauterine exposure to bacterial inoculation, though the physiological parameters of the fetus were relatively stable throughout the experimental protocol, even when challenged with intermittent hypoxic episodes. The placenta is an important mediator between mother and fetus during gestation, though its role in the inflammatory process is largely undefined. Studies on the placental role in the inflammatory process were undertaken, and the limited ability of proinflammatory cytokines and endotoxin to cross the placenta are detailed herein. Neurodevelopmental disorders can be monitored in animal models in order to determine effective disease models for characterization of injury and use in therapeutic strategies. Our characterizations of postnatal behaviour in the guinea pig model using motility monitoring and spatial memory testing have shown small but significant differences in pups exposed to inflammatory processes in utero. The data presented herein contributes a breadth of knowledge to the ongoing elucidation of the pathways by which fetal brain injury occurs. Determining the pathway of damage will lead to discovery of diagnostic criteria, while determining the vulnerabilities of the developing fetus is essential in formulating therapeutic options.

Fetal memory: Does it exist? what does it do?

Whether fetal memory exists has attracted interest for many thousands of years. The following review draws on recent experimental evidence to consider two questions: does the fetus have a memory? And, if so, what function(s) does it serve? Evidence from fetal learning paradigms of classical conditioning, habituation and exposure learning reveal that the fetus does have a memory. By comparison little attention has been paid to the possible function of memory. Possible functions discussed are: practice, recognition of and attachment to the mother, promotion of breastfeeding, and language acquisition. It is concluded that the fetus does possess a memory but that more attention to the functions of fetal memory will guide future studies of fetal memory abilities.

Sex differences in fetal habituation

There is some evidence for sex differences in habituation in the human fetus, but it is unknown whether this is due to differences in central processing (habituation) or in more peripheral processes, sensory or motor, involved in the response. This study examined whether the sex of the fetus influenced auditory habituation at 33weeks of gestation, and whether this was due to differences in habituation or in the sensory or motor components using a set of four experiments. The first experiment found that female fetuses required significantly fewer stimulus presentations to habituate than males. The second experiment revealed no difference in the spontaneous motor behaviour of male and female fetuses. The third experiment examined auditory intensity thresholds for the stimuli used to habituate the fetus. No differences in thresholds were found between males and females, although there was inter-individual variability in thresholds. A final experiment, using stimuli individualized for that particular fetus' auditory intensity threshold, found that female fetuses habituated faster than males. In combination, the studies reveal that habituation in the human fetus is affected by sex and this is due to a difference in central 'information processing' of the stimuli rather than peripheral aspects of the response. It is argued that male and female fetuses present different neurobehavioural developmental trajectories, with females more advanced at 33weeks than males. This study suggests that research examining prenatal behaviour should consider the factor of fetal sex. This may be particularly pertinent where there is an intention to use the results diagnostically. © 2012 Blackwell Publishing Ltd.

Fetal brain function in response to maternal alcohol consumption: Early evidence of damage

Background: Studies of the adverse neurobehavioral effects of maternal alcohol consumption on the fetus have been largely confined to the postnatal period, after exposure to alcohol has finished. This study explored the brain function of the fetus, at the time of exposure to alcohol, to examine its effect on information processing and stability of performance. Methods: Five groups of fetuses, defined by maternal alcohol consumption patterns, were examined: control (no alcohol); moderate (5 to 10 units/wk either drunk evenly across the week or as a binge, in 2 to 3 days); heavy (20+ units/wk drunk evenly or as a binge). Fetal habituation performance was examined on 3 occasions, separated by 7 days, beginning at 35 weeks of gestation. The number of trials required to habituate on each test session and the difference in performance across test sessions were recorded. Results: Fetuses exposed to heavy binge drinking required significantly more trials to habituate and exhibited a greater variability in performance across all test sessions than the other groups. Maternal drinking, either heavily but evenly or moderately as a binge, resulted in poorer habituation, and moderate binge drinking resulted in greater variability compared with no, or even, drinking. Conclusions: Decreased information processing, reflected by poorer habituation, and increased variability in performance may reflect the initial manifestations of structural damage caused by alcohol to the brain. These results will lead to a greater understanding of the effects of alcohol on the fetus's brain, enable the antenatal identification of fetal alcohol spectrum disorders, and lead to the early implementation of better management strategies. © 2012 by the Research Society on Alcoholism.


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Long-term flavor recognition in humans with prenatal garlic experience

The human fetus learns about its chemosensory environment and this influences its behavior at birth and during the nursing period. This study examined whether prenatal experience could influence behavior much later in life. The dietary preference of two groups of children (8- to 9-years old) was examined. Mothers of one group had consumed garlic during pregnancy, mothers of the control group had not. Children received two tests, 1 month apart, of a meal containing two portions of potato gratin, one flavored with garlic. The total amount of potato, and the percentage of garlic flavored potato, eaten was calculated and examined separately by ANOVA for factors of prenatal exposure, the child's sex, and trial. Children prenatally exposed to garlic ate significantly more garlic flavored potato and a significantly greater overall amount of potato on trial 2, compared to controls. The results demonstrate prenatal experience may affect behavior well into childhood. © 2012 Wiley Periodicals, Inc.

Cord blood erythropoietin, pH, PaO2 and haematocrit following caesarean section before labour

We have investigated the relationship between erythropoietin (Epo) and pH, PaO2 and haematocrit in 100 cord blood samples obtained at Caesarean section prior to labour. Of 82 term (> 37 weeks) infants, 64 were appropriately grown (10th-90th centiles), and their mean cord serum Epo and cord blood Epo was 23 +/- 8 mU/ml (mean +/- SD). Strong inverse correlations were found between cord serum Epo and cord blood pH (r = -0.74; p <0.0001), and between cord serum Epo and cord blood PaO2 (r = -0.55; p <0.0001), but not between cord serum Epo and cord haematocrit (r = 0.02; p <0.9). For the 18 preterm babies (gestation 32.4 +/- 4.1 weeks, birth weight 1,820 +/- 476 g), the Epo level was 36 +/- 8 mU/ml, which was not significantly greater than for the term babies. Strong inverse correlations were again found between Epo and pH (r = -0.87; p <0.0001) and Epo and PaO2 (r = -0.69; p <0.002). Babies from complicated pregnancies (intra-uterine growth retardation, pre-eclampsia, antepartum haemorrhage, diabetes mellitus and fetal distress) tended to have higher Epo levels. Thirteen babies had Epo levels > 40 mU/ml, and 11 (85%) of these required neonatal intensive care. Cord serum Epo correlates better with oxygen tension and pH at birth than with fetal growth and haematocrit, which are measures of chronic stress to the fetus.

A novel three-dimensional culture system for isolation and clonal propagation of neural stem cells using a thermo-reversible gelation polymer

In the present study, we examined the possible utility of a three-dimensional culture system using a thermo-reversible gelation polymer to isolate and expand neural stem cells (NSCs). The polymer is a synthetic biologically inert polymer and gelates at temperatures higher than the gel-sol transition point ( approximately 20 degrees C). When fetal mouse brain cells were inoculated into the gel, spherical colonies were formed ( approximately 1% in primary culture and approximately 9% in passage cultures). The spheroid-forming cells were positive for expression of the NSC markers nestin and Musashi. Under conditions facilitating spontaneous neural differentiation, the spheroid-forming cells expressed genes characteristic to astrocytes, oligodendrocytes, and neurons. The cells could be successively propagated at least to 80 poly-D-lysines over a period of 20 weeks in the gel culture with a growth rate higher than that observed in suspension culture. The spheroids formed by fetal mouse brain cells in the gel were shown to be of clonal origin. These results indicate that the spheroid culture system is a convenient and powerful tool for isolation and clonal expansion of NSCs in vitro.

The Role of Growth Trajectories in Classifying Fetal Growth Restriction

OBJECTIVE: To examine the validity of a growth trajectory method to discriminate between pathologically and constitutionally undergrown fetuses using repeated measures of estimated fetal weight.

METHODS: In a prospective, observational, multicenter study in Ireland, 1,116 women with a growth-restricted fetus diagnosed participated with the objective of evaluating ultrasound findings as predictors of pediatric morbidity and mortality. Fetal growth trajectories were based on estimated fetal weight.

RESULTS: Between 22 weeks of gestation and term, two fetal growth trajectories were identified: normal (96.7%) and pathologic (3.3%). Compared with the normal trajectory, the pathologic trajectory was associated with an increased risk for preeclampsia (odds ratio [OR] 8.1, 95% confidence interval [CI] 2.6–23.4), increased umbilical artery resistance at 30 weeks of gestation (OR 12.6, 95% CI 4.6–34.1) or 34 weeks of gestation (OR 28.0, 95% CI 8.9–87.7), reduced middle cerebral artery resistance at 30 weeks of gestation (OR 0.33, 95% CI 0.12–0.96) or 34 weeks of gestation (OR 0.14, 95% CI 0.03–0.74), lower gestational age at delivery (mean 32.02 weeks of gestation compared with 38.02 weeks of gestation; P<.001), and higher perinatal complications (OR 21.5, 95% CI 10.5–44.2). In addition, 89.2% of newborns with pathologic fetal growth were admitted to neonatal intensive care units compared with 25.9% of those with normal growth.

CONCLUSIONS: Fetal growth trajectory analysis reliably differentiated fetuses with a pathologic growth pattern among a group of women with growth-restricted fetuses. With further development, this approach could provide clarity to how we define, identify, and ultimately manage pathologic fetal growth.

LEVEL OF EVIDENCE: II

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Background: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown.

Methods: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses.

Results: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1).

Conclusions: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.