S.W. D'Souza

Amino acid (system A) transporter activity in microvillous membrane vesicles from the placentas of appropriate and small for gestational age babies

ABSTRACT: Although a number of causes of poor fetal growth are known, the involvement of placental transport proteins in the etiology of growth retardation is not understood. The aim of this study was to investigate the activity of the system A amino acid transporter and the Na+/H+ exchanger in vesicles isolated from the microvillous membrane of the syncytiotrophoblast of placentas of appropriate and small for gestational age babies. There were no biochemical differences between the membranes from the two groups of placentas, and there was no difference in the activity of the Na+/H+ exchanger. The initial rate of uptake of methylaminoisobutyric acid (a nonmetabolizable amino acid analogue) was 63% lower in vesicles from placentas of small for gestational age babies. Kinetic analysis of the system A transporter (utilized by methylaminoisobutyric acid) showed that the Vmax in the vesicles from placentas of small for gestational age babies (0.24 ± 0.03 nmol/mg protein/30 s, n = 5) was significantly lower than that in vesicles from placentas of appropriate for gestational age babies (0.64 ± 0.09 nmol/mg protein/30 s, n = 4, p < 0.001), whereas the Km was not different between the two groups. It is concluded that there is an abnormality of system A amino acid transporter function in placentas of small for gestational age babies.

Placental Phenotypes of Intrauterine Growth

The placenta is essential to nutrition before birth. Recent work has shown that a range of clearly defined alterations can be found in the placentas of infants with intrauterine growth restriction (IUGR). In the mouse, a placental specific knockout of a single imprinted gene, encoding IGF-2, results in one pattern of alterations in placenta structure and function which leads to IUGR. We speculate that the alterations in the human placenta can also be grouped into patterns, or phenotypes, that are associated with specific patterns of fetal growth. Identifying the placental phenotypes of different fetal growth patterns will improve the ability of clinicians to recognize high-risk patients, of laboratory scientists to disentangle the complexities of IUGR, and of public health teams to target interventions aimed at ameliorating the long-term adverse effects of inadequate intrauterine growth.

Increased survival and deteriorating developmental outcome in 23 to 25 week old gestation infants, 1990–4 compared with 1984–9

AIMS To assess whether changes in survival over time in infants of 23 to 25 weeks of gestational age were accompanied by changes in the incidence of disability in childhood during an 11 year period. METHODS Obstetric and neonatal variables having the strongest association with both survival to discharge from a regional neonatal medical unit and neurodevelopmental disability in 192 infants of 23 to 25 weeks of gestation, born in 1984 to 1994, were studied as a group and in two cohorts (1984 to 1989 n = 96 and 1990 to 1994 n = 96). The data collected included CRIB (clinical risk index for babies) scores and cranial ultrasound scan findings. The children were followed up at outpatient clinics. RESULTS Between 1984 and 1989 (cohort 1) and 1990 and 1994 (cohort 2) the rate of survival to discharge increased significantly from 27% to 42% and the rate of disability in survivors increased from 38% to 68% ; most of this increase was in mild disability. The proportions of survivors with cerebral palsy did not alter significantly (21% vs 18%), but more survivors with blindness due to retinopathy of prematurity (4% vs18%), myopia (4% vs 15%) and squints (8%vs 13%) contributed to the increased rate of disability. Clinically significant cranial ultrasound findings and a high CRIB score were strongly associated with death. A high CRIB score was most strongly associated with disability. CONCLUSIONS The rise in disability with improved survival was not due to cerebral palsy; rather the main contributors were blindness due to retinopathy, myopia, and squint. The causes of these disabilities seem to be linked to high CRIB scores. A system of regular and skilled retinal examination and access to facilities for retinal ablation should be in place in all neonatal units which undertake the care of such extremely preterm infants.

Expression of folate transporters in human placenta and implications for homocysteine metabolism

Poor folate status during pregnancy can lead to elevated maternal plasma levels of homocysteine (Hcy) with associated pregnancy complications and adverse neonatal outcomes, suggesting placental metabolism of Hcy might be an important determinant in influencing fetal development. The metabolic pathways for Hcy in placenta are not well defined. In this study we examined the gene expression of key enzymes involved in Hcy metabolism in first trimester and term human placenta to determine which metabolic pathways prevail. Expression of mRNA for methionine synthase and 5,10-methylene tetrahydrofolate reductase, enzymes involved in the methionine cycle and responsible for the re-methylation of Hcy to methionine, were expressed at similar levels between first trimester and term and in comparison to human liver as positive control. In contrast, cystathionine beta-synthase mRNA expression was markedly lower than that in liver at both gestational periods. Betaine-homocysteine methyltransferase mRNA was undetectable at either gestational age. These data suggest that re-methylation of Hcy using methyl donation from 5-methyltetrahydrofolate is the prevalent pathway, indicating a marked reliance on folate availability. This led to further investigations examining the expression and localisation of folate transporters in first trimester and term placenta. Folate receptor alpha (FRalpha) was highly polarised to the microvillous plasma membrane (MVM) of the syncytiotrophoblast at both gestational periods, a distribution shared by the proton-coupled folate transporter which co-localised with FRalpha. Reduced folate carrier was distributed to both MVM and basal syncytiotrophoblast plasma membranes at term suggesting a role at both loci, and in first trimester was localised to MVM as well as cytotrophoblast plasma membranes. These data support the concept that placental folate transport is established early in pregnancy, providing folate for utilisation in placental Hcy metabolism.

Altered activity of the system A amino acid transporter in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women.

Fetal macrosomia (FM) is a well-recognized complication of diabetic pregnancy but it is not known whether placental transport mechanisms are altered. We therefore studied the activity of the system A amino acid transporter, the system L amino acid transporter, and the Na+/H+ exchanger in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women (FM group), from placentas of appropriately grown babies born to diabetic women (appropriate for gestational age group) and from placentas of appropriately grown babies of normal women (control group). Sodium-dependent uptake of [14C]-methylaminoisobutyric acid at 30 s (initial rate, a measure of system A activity) was 49% lower into FM vesicles than into control vesicles (P < 0.02); this effect was due to a decrease in Vmax of the transporter with no change in Km. There was no significant difference in system A activity between the appropriate for gestational age group and control or FM group. There was also no difference between system L transporter or Na+/H+ exchanger activity between the three groups. We conclude that the number of system A transporters per milligram of membrane protein in the placental microvillous membrane is selectively reduced in diabetic pregnancies associated with FM.

Placental transporter activity and expression in relation to fetal growth

The question of whether there are causative or compensatory changes in placental transport physiology affecting fetal growth is considered. Reductions in uterine and umbilical blood flow in growth retardation will reduce maternofetal exchange of lipophilic solutes, such as O2 and CO2, but will not have a major effect on the transfer of hydrophilic solutes. These solutes are transferred across the placenta by paracellular diffusion, transporter protein‐mediated transport and endocytosis‐exocytosis. Neither paracellular diffusion nor endocytosis‐exocytosis has been investigated in relation to fetal growth. The weight of evidence is that there is no change in the activity and expression of the syncytiotrophoblast GI UTI glucose transporter in fetal growth retardation. However, there is strong evidence that the activity of the system A amino acid transporter, per milligram of placental membrane protein, is altered in relation to fetal growth, but in a complex manner. There is also some weaker evidence that the activity of the Na(+)‐H+ exchanger, per milligram of placental membrane protein, is directly related to birth‐weight. There are no data for other solute transporters; a considerable amount of work still remains to be done in order to understand the relationship between placental function and fetal growth rate.

Postnatal changes in N‐methyl‐D‐aspartate receptor binding and stimulation by glutamate and glycine of [3H]‐MK‐801 binding in human temporal cortex

1 Homogenates of human infant and adult temporal cortex were used to measure [3H]‐TCP and [3H]‐MK‐801 binding to the N‐methyl‐d‐aspartate (NMDA)‐coupled ion channel phencyclidine site. 2 Both [3H]‐TCP and [3H]‐MK‐801 binding increased in infant cortex by> 100% between term and 26 weeks suggesting that the numbers of NMDA receptors increase during postnatal brain development. 3 [3H]‐MK‐801 binding was measured under non‐equilibrium conditions in temporal cortex homogenates with the addition of 100 μm of l‐glutamate plus a range of concentrations (0.05 μm−100 μm) of glycine. Glutamate and glycine increased [3H]‐MK‐801 binding by stimulating NMDA receptors and improving [3H]‐MK‐801 access to ion channel binding sites; maximum stimulation in adult and infant temporal cortex was achieved with 100 μm glutamate plus 5 μm glycine; a higher concentration of glycine (50 μm) reduced [3H]‐MK‐801 binding to below maximum. 4 The stimulation by 100 μm glutamate plus 5 μm glycine of [3H]‐MK‐801 binding in infant temporal cortex was affected by postnatal age. For example, although the stimulation of [3H]‐MK‐801 binding in 5–6 week infant cortex (236% of basal) was similar to adult cortex (230% of basal), in samples taken from infants aged 5–6 months glycine (plus glutamate) stimulation of [3H]‐MK‐801 binding (392% of basal) was substantially greater than that measured in adult temporal cortex. 5 The binding of [3H]‐glycine to the glycine modulatory site associated with the NMDA receptor in infant cortex also increased with postnatal age by > 100% between term and 26 weeks. 6 It is concluded that NMDA receptors in infant cortex increase to levels greater than those in adult cortex during postnatal development. The results do not exclude the possibility that the transiently increased NMDA receptor‐ion channel complex in infant cortex shows enhanced responses to agonists and modulators.

Children conceived by in vitro fertilisation after fresh embryo transfer

AIMS To compare the outcome in in vitro fertilisation (IVF) children (after fresh embryo transfer) from multiple and singleton births with one another, and with normally conceived control children. METHODS A cohort of 278 children (150 singletons, 100 twins, 24 triplets and four quadruplets), conceived by IVF after three fresh embryos had been transferred, born between October 1984 and December 1991, and 278 normally conceived control children (all singletons), were followed up for four years after birth. They were assessed for neonatal conditions, minor congenital anomalies, major congenital malformations, cerebral palsy and other disabilities. Control children, all born at term, were matched for age, sex and social class. RESULTS The ratio of male:female births was 1.03. Forty six per cent of IVF children were from multiple births; 34.9% were from preterm deliveries; and 43.2% weighed less than 2500 g at birth. The IVF singletons were on average born one week earlier than the controls, weighed 400 g less, and had a threefold greater chance of being born by caesarean section. The higher percentage of preterm deliveries was largely due to multiple births and they contributed to neonatal conditions in 45.0% of all IVF children. The types of congenital abnormalities varied: 3.6% of IVF children and 2.5% of controls had minor congenital anomalies, and 2.5% of IVF children and none of the controls had major congenital malformations. The numbers of each specific type of congenital abnormality were small and were not significantly related to multiple births. IVF children (2.1%) and 0.4% of the controls had mild/moderate disabilities. They were all from multiple births, including two children with cerebral palsy who were triplets. CONCLUSIONS The outcome of IVF treatment leading to multiple births is less satisfactory than that in singletons because of neonatal conditions associated with preterm delivery and disabilities in later childhood. A reduction of multiple pregnancies by limiting the transfer of embryos to two instead of three remains a high priority.

Modulation by magnesium of N-methyl-D-aspartate receptors in developing human brain

AIM To investigate age related alterations in glutamate N-methyl-D-aspartate (NMDA) receptor binding produced by the modulatory compounds glutamate, glycine, and magnesium (Mg2+) sulphate. METHODS The effects produced by glutamate plus glycine, and Mg2+ on the binding of [3H]MK-801, a ligand for theN-methyl-D-aspartate ion channel phencyclidine site, were measured in membrane preparations made from prefrontal cortex from human neonate (n = 5), infant (n = 6), and adult (n = 6) necropsy brains. RESULTS Neonatal brains had the least [3H]MK-801 binding, suggesting either a low density of NMDA receptors or a more restricted access of [3H]MK-801 to cation channel sites. Infant brains had the most [3H]MK-801 binding which was stimulated to a greater extent by L-glutamate (100 μM) and glycine (10 μM) than in neonatal and adult brains. Mg2+ invariably inhibited [3H]MK-801 binding. However, the Mg2+IC50 value was higher in neonatal brain (3.6 mM) than infant (1.4 mM) and adult (0.87 mM) brains. CONCLUSION Infant brain may have excess NMDA receptors which are hyper responsive to glutamate and glycine. The lower potency of Mg2+ to inhibit [3H]MK-801 binding in neonatal cortex may be because newborn babies have NMDA receptors without the normal complement of Mg2+ sites. The findings suggest that therapeutic NMDA receptor block in neonates requires higher concentrations of magnesium sulphate in brain tissue.

Antiepileptic Medication During Pregnancy: Does Fetal Genotype Affect Outcome?

Congenital abnormalities and impaired development in childhood are attributable to fetal exposure to antiepileptic drugs (AEDs). Pregnancy registries set up to obtain information about the potential risks of fetal exposure to AEDs, in particular major congenital malformations (MCMs), suggest that valproate exposure increases the frequency of congenital malformations more than other AEDs. Furthermore, follow-up studies have drawn attention to cognitive impairments in later childhood after prenatal exposure to valproate. Fetal exposure to AEDs may be influenced by drug transporting proteins in the placenta, including P-glycoprotein (P-gp), multidrug resistance protein (MRP) 1, and breast cancer resistance protein (BCRP). Their location in the syncytiotrophoblast plasma membrane, at the interface of the maternal and fetal circulations, allows these transport proteins to efflux xenobiotics back to the mother and offers the fetus protection from medications taken during pregnancy. Genetic variations in the expression and activity of these transport proteins may influence fetal exposure to AEDs and thus the risk of teratogenicity. Identification of a hierarchy of haplotypes ranging from susceptible to protective of congenital abnormalities could assist genetic counseling, in assessing fetal risks from exposure to AEDs.