Quantification of plasma myo-inositol using gas chromatography-mass spectrometry.

BACKGROUND: Myo-inositol (MI) deficiency is associated with an increased risk for neural tube defects (NTDs), mental disorders and metabolic diseases. We developed a gas chromatography-mass spectrometry (GC-MS) method to detect MI in human plasma, which was accurate, relatively efficient and convenient for clinical application. METHODS: An external standard method was used for determination of plasma MI. Samples were analyzed by GC-MS after derivatization. The stable-isotope labeled internal standard approach was used to validate the method's accuracy. Alpha fetal protein (AFP) was detected by chemiluminescence immunoassay. RESULTS: The method was validated by determining the linearity, sensitivity and recovery rate. There was a good agreement between the internal standard approach and the present method. The NTD-affected pregnancies showed lower plasma MI (P=0.024) and higher AFP levels (P=0.001) than control. Maternal MI level showed a better discrimination in spina bifida subgroup, while AFP level showed a better discrimination in anencephaly subgroup after stratification analysis. CONCLUSIONS: We developed a sensitive and reliable method for the detection of clinical plasma MI, which might be a marker for NTDs screening, and established fundamental knowledge for clinical diagnosis and prevention for the diseases related to disturbed MI metabolism.

Is low iron status a risk factor for neural tube defects?

BACKGROUND: Folic acid supplements can protect against neural tube defects (NTDs). Low folate and low vitamin B12 status may be maternal risk factors for having an NTD affected pregnancy. However, not all NTDs are preventable by having an adequate folate/ B12 status and other potentially modifiable factors may be involved. Folate and vitamin B12 status have important links to iron metabolism. Animal studies support an association between poor iron status and NTDs, but human data are scarce. We examined the relevance of low iron status in a nested NTD case-control study of women within a pregnant population-based cohort. METHODS: Pregnant women were recruited between 1986 and 1990, when vitamin or iron supplementation in early pregnancy was rare. Blood samples, taken at an average of 14 weeks gestation, were used to measure ferritin and hemoglobin in 64 women during an NTD affected pregnancy and 207 women with unaffected pregnancies. RESULTS: No significant differences in maternal ferritin or hemoglobin concentrations were observed between NTD affected and nonaffected pregnancies (case median ferritin 16.9 µg/L and hemoglobin 12.4 g/dl versus 15.4 µg/L and 12.3g/dl in controls). As reported previously, red cell folate and vitamin B12 concentrations were significantly lower in cases. Furthermore, there was no significant association of iron status with type of NTD lesion (anencephaly or spina bifida). CONCLUSION: We conclude that low maternal iron status during early pregnancy is not an independent risk factor for NTDs. Adding iron to folic acid for periconceptional use may improve iron status but is not likely to prevent NTDs.

Impact of folate supplementation on the efficacy of sulfadoxine/pyrimethamine in preventing malaria in pregnancy: the potential of 5-methyl-tetrahydrofolate.

Malaria remains the leading cause of mortality and morbidity in children under the age of 5 years and pregnant women. To counterbalance the malaria burden in pregnancy, an intermittent preventive treatment strategy has been developed. This is based on the use of the antifolate sulfadoxine/pyrimethamine, taken at specified intervals during pregnancy, and reports show that this approach reduces the malaria burden in pregnancy. Pregnancy is also associated with the risk of neural tube defects (NTDs), especially in women with low folate status, and folic acid supplementation is recommended in pregnancy to lower the risk of NTDs. Thus, in malaria-endemic areas, pregnant women have to take both antifolate medication to prevent malaria and folic acid to lower the risk of NTDs. However, the concomitant use of folate and antifolate is associated with a decrease in antifolate efficacy, exposing pregnant women to malaria. Thus, there is genuine concern that this strategy may not be appropriate. We have reviewed work carried out on malaria folate metabolism and antifolate efficacy in the context of folate supplementation. This review shows that: (i) the folate supplementation effect on antifolate efficacy is dose-dependent, and folic acid doses required to protect pregnant women from NTDs will not decrease antifolate activity; and (ii) 5-methyl-tetrahydrofolate, the predominant form of folate in the blood circulation, could be administered (even at high dose) concomitantly with antifolate without affecting antifolate efficacy. Thus, strategies exist to protect pregnant women from malaria while maintaining adequate folate levels in the body to reduce the occurrence of NTDs.

Serological differences in folate/vitamin B12 in pregnancies affected by neural tube defects.

BACKGROUND: Laboratory evidence is presented of significant associations between reduced maternal serum folate and vitamin B12 levels and neural tube birth defects (NTD) compared to referents. METHODS: This was an incident case-control study. Cases of neural tube defects (including anencephaly and open spina bifida) diagnosed in residents within 100 miles of the US-Mexico border from January 1993 to October 2000 were eligible. Most cases were diagnosed in utero upon visits to clinics, obstetrical or genetic expert offices. Cases identified upon hospital admission or at delivery were also eligible. Cases identified after discharge were not. Controls were matched on geographic region, maternal age, race/ethnicity, gestational age, and type of health insurance (including none). RESULTS: Three hundred eighty-two border area residents (107 cases and 275 individually matched controls) provided biological specimens. Median folate concentrations for case mothers were 36% lower than controls (9.8 ng/mL vs. 15 ng/mL). Maternal serum folate concentrations in quartiles above 9.5 ng/mL indicated significantly reduced risk (OR = 0.4, OR = 0.3, and OR = 0.2). Likewise, the risk for NTD decreased (OR = 0.4, OR = 0.3, and OR = 0.2) in quartiles of sera B12 concentrations above 246 pg/mL. CONCLUSIONS: Physician attention is invited to significantly lower concentrations of serum folate and vitamin B12 in women with NTD-affected pregnancies. This study assayed sera samples from women while still pregnant or immediately after delivery. The confounding effect of reduced folate and B12 levels with other biological and chemical exposures will be addressed in subsequent communications.

Second trimester levels of maternal serum inhibin A in pregnancies affected by fetal neural tube defects.

Inhibin A is effective as a second trimester maternal serum marker for Down syndrome screening. In the present study, inhibin A levels were measured in second trimester maternal serum samples from 28 pregnancies affected with open neural tube defects; 12 associated with open spina bifida and 16 associated with anencephaly. Each measurement was expressed as a multiple of the median (MoM) for control singleton pregnancies (n=1464) of the same completed week of gestation. Inhibin A levels were not significantly altered in cases of open neural tube defects; the median value was 0.96 MoM in cases of open spina bifida and 1.19 MoM in cases of anencephaly. Therefore, second trimester maternal serum inhibin A levels will not have an impact on prenatal detection of open neural tube defects.

Combination of elevated maternal serum alpha-fetoprotein (MSAFP) and low estriol is highly predictive of anencephaly.

Increased levels of second trimester maternal serum alpha-fetoprotein (MSAFP) have long been established as a marker for neural tube defects (NTDs). In addition, decreased levels of maternal estriol in the third trimester have been reported in pregnancies with anencephalic fetuses. The purpose of this study was to evaluate whether early second trimester unconjugated serum estriol (uE3) is an independent predictor of NTDs. The study included 57,031 patients who underwent maternal serum screening with MSAFP at 14-22 weeks gestation. Of these, 23,415 also had uE3 measurements. There were 63 cases of NTD, an overall incidence of 1.1 per 1,000. Elevated MSAFP (> or =2.5 MOM) was detected in 1,346 patients, 48 of which had NTDs. Decreased uE3 (< or =0.5) was detected in 1,437 patients, 17 of which had NTDs. The incidence of NTDs was significantly higher in patients with low uE3, compared to patients with normal/high uE3 (1.15% vs. 0.09%, P < 001). Finally, 51 patients had both increased MSAFP and decreased uE3; 16 of these had NTDs, 14 of which were anencephalics. In conclusion, both elevated MSAFP and low maternal serum estriol are predictive of NTD but have a low sensitivity. The combination of abnormally elevated MSAFP and low estriol is highly predictive of NTD in particular anencephaly.

Maternal serum alpha-fetoprotein in fetal neural tube and abdominal wall defects at 10 to 14 weeks of gestation.

Maternal serum alpha-fetoprotein concentration was determined in nine pregnancies with fetal anencephaly, seven with exomphalos containing liver, two with spina bifida and 100 normal controls at 10 to 14 weeks of gestation. The median alpha-fetoprotein in the group with fetal anencephaly and exomphalos was significantly higher than in normal fetuses but the sensitivity of this test is likely to be only about 30% for a false positive rate of 5%.

Maternal and fetal serum and red blood cell folate and vitamin B12 concentrations in pregnancies affected by neural tube defects.

In the present study maternal and fetal serum and red cell folate and vitamin B12 concentrations were determined in NTD affected and in control pregnancies. Fetal serum folate (p < 0.001) and red cell folate (p < 0.001) concentrations were significantly higher than maternal levels in the cases as well as in the controls. Maternal and fetal vitamin B12 concentrations did not differ significantly in both groups. There were also no significant differences in maternal and fetal vitamin concentrations in the peripheral blood between cases and controls. However, when a subgroup analysis was performed according to the type of NTD we found significant differences in fetal vitamin B12 status between anencephaly and spina bifida cases as well as between anencephaly cases and the controls.

Circulating levels of growth hormone, insulin-like growth factor-I and prolactin in normal, growth retarded and anencephalic human fetuses.

We measured growth hormone (GH), insulin-like growth factor-I (IGF-I), and both total and glycosylated prolactin (PRL) levels in 131 blood samples obtained by cordocentesis in normal and abnormal fetuses from 19 to 40 weeks of gestation. In normal fetuses, IGF-I and PRL levels showed a positive correlation and GH a negative correlation with gestational age. A negative relation between GH and IGF-I levels was observed, while PRL did not show any correlation with both GH and IGF-I concentrations. IGF-I increased from 5.6 +/- 3 (at 19-22 weeks) to 10.7 +/- 5 nmol/l at term; GH decreased from 31 +/- 10 to 7.7 +/- 4 micrograms/l and PRL increased from 16 +/- 18 to 139 +/- 76 micrograms/l. Glycosylated PRL accounted for about 15% of total PRL, a value similar to that found in normal adults. In 27 fetuses of 27-37 weeks with intra-uterine growth retardation, GH and PRL levels were higher and IGF-I levels lower than in normal fetuses matched for week of gestation. In 8 anencephalic fetuses of 19-26 weeks of gestation, both GH and IGF-I levels were lower, and PRL levels were higher than in matched controls. Altogether these data support the views that a) both GH and PRL secretion are under the hypothalamic control during fetal development, b) the serum GH decrease from midgestation to the end of pregnancy is mediated by the negative feed-back mechanism of increasing IGF-I levels and c) IGF-I production is mainly regulated by fuel supply and only partially by GH.

[Noninvasive serum test for prenatal detection of Down syndrome, other chromosome abnormalities and open neural tube defects--a prospective study]. / Nichtinvasiver Serum-Test (NIS) zur pränatalen Erfassung von Morbus Down, anderen chromosomalen Anomalien und offenen Neuralrohrdefekten--Eine prospektive Studie.

Between September 1st 1990 and Juli 31st 1993, 5071 pregnant women were screened prospectively by the "triple-test", including maternal serum alpha-fetoprotein, human chorionic gonadotropin and unconjugated oestriol in order to detect chromosomal anomalies and open neural tube defects. The serum samples were collected in collaboration with the obstetricians of the region of West-Mecklenburg and North-West-Brandenburg. Laboratory testing using radioimmunoassays was performed between weeks 15 and 20 of gestation, all serum specimens being investigated in only one institution. The original alpha-software from Wald et al. was the basis for calculating the statistical risk for Down's syndrome. Pregnant women with a high risk for Down's syndrome (cutoff > or = 1:250) were taken care of in a special outpatient clinic including procedures like amniocentesis and fetal blood sampling. Amongst 5071 pregnant women, 21 fetal anomalies were seen. Five cases of Down's syndrome, three of trisomy 18, one trisomy 13, two cases of triploidy and four cases of open neural tube defects, one 46 xy/45 x mosaic karyotype and one case of gastroschisis could be diagnosed correctly. One case of trisomy 21, one case of trisomy 18 and two open neural tube defects showed false negative results. Using the cutoff of 1:250 for prenatal detection of Down's syndrome and performing ultrasound routinely to determine gestational age, the sensitivity of the "triple-test" was 83.33% having a specificity of 92.68%. The predictive value of a positive test for prenatal diagnosis of Down's syndrome was 1.33%.(ABSTRACT TRUNCATED AT 250 WORDS)

[2 cases of anencephaly in one family]. / Dwa przypadki bezmózgowia w jednej rodzinie.

Two births of anencephalic fetuses occurred in the second and the fourth pregnancies in a family. The children born after the 1st and 3rd pregnancies were normal. Anencephaly was not associated with spina bifida, and both fetuses were female. The study failed to demonstrate the cause of the abnormality.

Quick measurement of serum unconjugated estrogens, and its clinical application to abnormal pregnancies and ovarian follicle maturation.

Using an automated HPLC system reported recently by us (J Clin Lab Anal 4:410-413, 1990), serum unconjugated estriol and estradiol (u-E3 and u-E2) can be measured within 22 min. The clinical value of this method was evaluated in this study with abnormal pregnancies, twin pregnancies, and pregnancies associated with fetal anencephaly or complicated by gestational toxicosis or diabetes mellitus. A significant correlation was demonstrated between an abrupt decrease in serum u-E3 level and an abnormal pattern on fetal heart rate monitor. Since urinary E3 was not sensitive enough to detect any depressed fetal condition, serum u-E3 is better biochemical parameter for assessment of fetal well-being. In addition, u-E2 was measured in the sera of nonpregnant women with separation chromatography as a pretreatment in order to obtain higher sensitivity of the system. This modified HPLC system enabled us to determine the serum u-E2 in about 60 min with the minimal detectable value of 80 pg/ml; the coefficient of correlation with RIA was 0.822. As a result, this quick measurement of u-E3 and u-E2 by our HPLC system revealed to be useful not only for evaluating fetoplacental function but also for monitoring ovarian follicle maturation.

Fetal hyperinsulinism in anencephaly.

Insulin was measured in the blood collected in utero from three midtrimester anencephalic fetuses. The hyperinsulinism found could be due to an underutilization of glucose in the absence of most of the brain and could be responsible for the relatively normal growth in anencephaly despite the absence of the hypothalamohypophysial axis.

The effects of dexamethasone and anencephaly on newborn serum levels of apolipoprotein A-1.

Since the control of production and clearance of plasma lipoproteins in utero is largely unknown, we sought to evaluate the effects of glucocorticosteroid (dexamethasone) treatment of developing fetuses and of chronic intrauterine hypercholesterolemia, due to fetal anencephaly, on newborn serum levels of apolipoprotein A-1 (Apo A-1), the major apoprotein of high density lipoproteins (HDL). Among preterm newborn infants (26-32 weeks gestation), the total, HDL, and low density lipoprotein cholesterol levels in umbilical cord serum of 11 newborns exposed to 4 doses of dexamethasone (5 mg each) within 1 week of delivery [mean, 3.05 +/- 1.01 (+/- SD), 0.83 +/- 0.18, and 1.84 +/- 0.69 mmol/L, respectively] and of 3 anencephalic newborns (2.84 +/- 0.57, 0.83 +/- 0.36, and 1.89 +/- 0.54 mmol/L) were increased to a similar extent over those in 17 normal newborns (1.76 +/- 0.16, 0.62 +/- 0.16, and 1.14 +/- 0.13 mmol/L). On the other hand, umbilical cord serum Apo A-1 levels were markedly increased only in the dexamethasone-treated preterm newborns (1.35 +/- 0.55 g/L; anencephalic, 0.78 +/- 0.15 g/L; normal, 0.68 +/- 0.06 g/L). Also, whereas serum total, HDL, and low density lipoprotein cholesterol levels in 5 term anencephalic newborns (3.85 +/- 1.37, 1.06 +/- 0.08, and 2.64 +/- 0.91 mmol/L) were substantially higher than those in 41 normal term newborns (1.42 +/- 0.28, 0.57 +/- 0.12, and 0.74 +/- 0.05 mmol/L), serum Apo A-1 levels were similar at term in anencephalic (1.01 +/- 0.30 g/L) and normal newborns (0.99 +/- 0.08 g/L). Normal Apo A-1 and lipoprotein cholesterol levels were found in an additional newborn who was delivered 30 days after exposure to dexamethasone. We conclude that intrauterine glucocorticosteroid treatment leads to transiently increased serum Apo A-1 levels in the newborn. This increase, however, is not likely to be secondary consequence of the hypercholesterolemia that also occurs in such newborns, since hypercholesterolemia of a similar extent in anencephalic newborns, who have atrophic adrenals, was not associated with marked changes in serum Apo A-1 levels.

Mineralocorticosteroids and pregnancy: regulation of extra-adrenal deoxycorticosterone production by estrogen.

During pregnancy, deoxycorticosterone (DOC) in maternal plasma is produced principally by 21-hydroxylation of circulating progesterone in a number of extra-adrenal tissues. Estrogen acts, directly or indirectly, to increase the transfer constant of conversion of progesterone to DOC. In this study, we evaluated the regulation of DOC production during pregnancy by considering the plasma levels of progesterone, the kinetics of extra-adrenal steroid 21-hydroxylase, and the stimulation of the conversion of progesterone to DOC by estrogen.

Lecithin-sphingomyelin ratios in amniotic fluid of pregnancies with an anencephalic fetus.

As many investigators have shown that surfactant production in the developing human lung is subject to multihormonal regulation, the present authors determined the lecithin-sphingomyelin (L/S) ratio in amniotic fluid of pregnancies with an anencephalic fetus, in which there was known to be aberrant production of fetal pituitary, adrenal, and consequently, placental hormones. The L/S ratio in amniotic fluid from seven of eight pregnancies with an anencephalic fetus was substantially lower than that in amniotic fluids of pregnancies with a normal fetus at the same stage of gestation. The L/S ratio in amniotic fluid of an anencephalic fetus of a twin pregnancy (monochorionic diamniotic) at 34 weeks' gestation was low; the L/S ratio of the amniotic fluid of the normal fetus was high. These data are supportive of the view that fetal lung maturation is dependent, in part, upon normal function of the fetal pituitary and adrenal.

Insulin-like growth factors (IGF) 1 and 2 in human foetal plasma and relationship to gestational age and foetal size during midpregnancy.

IGF-1 and IGF-2 were measured by specific radioimmunoassay after acid-ethanol extraction of plasma obtained by foetoscopy from 20 normal foetuses aged 15-23 weeks. IGF-1 and IGF-2 levels were 36 +/- 11 and 162 +/- 55 ng/ml, respectively. In comparison, levels in cord blood were 84 +/- 58 and 264 +/- 176 ng/ml, respectively, and in adult plasma were 410 +/- 106 and 818 +/- 272 ng/ml. Both IGF-1 and IGF-2 were in the normal foetal range in a further three foetuses with anencephaly and two foetuses with spina bifida. No sex difference was observed. IGF-1 was positively correlated with foetal body weight (P less than 0.001), placenta weight (P less than 0.02) and with body length measured crown-rump (P less than 0.01) or crown-heel (P less than 0.02). No correlation between IGF-2 and body weight, length, placenta weight or gestational age was found. Both IGF-1 and IGF-2 are present in the human foetal circulation earlier in gestation than has previously been demonstrated, the levels being low throughout this period of gestation in comparison with adult plasma.

Umbilical cord plasma concentrations of deoxycorticosterone sulfate in anencephalic fetuses.

In the present investigation, we determined the levels of deoxycorticosterone sulfate in mixed umbilical cord plasma of anencephalic abortuses and newborn infants. The anencephalic fetus is an interesting model with respect to the production of deoxycorticosterone and deoxycorticosterone sulfate on several accounts. There is profound adrenal atrophy in most such fesuses, and, in consequence, there also is relatively profound hypoestrogenism. This is an important consideration in the formation of deoxycorticosterone and deoxycorticosterone sulfate since it is known that estrogen acts to stimulate extra-adrenal steroid 21-hydroxylase and 21-hydroxysteroid sulfotransferase activities. The plasma levels of deoxycorticosterone sulfate in 22 anencephalic abortuses and newborn infants delivered between 21.5 and 45.5 weeks of gestation ranged from 1.8 to 30.3 ng/ml. The concentrations of deoxycorticosterone sulfate in umbilical cord plasma of anencephalic fetuses and newborn infants were not related to gestational age or method of delivery and, at term, were less than 13% of those in umbilical cord plasma of normal newborn infants. These data can be interpreted to indicate (1) that deoxycorticosterone sulfate normally is secreted directly by the fetal adrenal or (2) that placental estrogen normally derived largely from fetal adrenal dehydroisoandrosterone sulfate is essential for the maintenance of plasma deoxycorticosterone sulfate levels in the fetus by stimulating extra-adrenal deoxycorticosterone sulfate production from plasma progesterone, or both.

Serum copper concentration significantly less in abnormal pregnancies.

We estimated copper concentration in maternal serum during 244 normal and 15 abnormal pregnancies. Values were lower in the abnormal pregnancies than in the normal ones, and did not vary with gestational age between 15 to 18 weeks in normal pregnancies.