Docosahexaenoic acid (DHA) intake estimated from a 7-question survey identifies pregnancies most likely to benefit from high-dose DHA supplementation.

BACKGROUND: Two randomized trials found women with low blood docosahexaenoic acid (DHA; an omega 3 fatty acid) had fewer early preterm births (<34 weeks gestation) if they were assigned to high dose DHA supplementation, however, there is currently no capacity for clinicians who care for pregnancies to obtain a blood assessment of DHA. Determining a way to identify women with low DHA intake whose risk could be lowered by high dose DHA supplementation is desired. OBJECTIVE: To determine if assessing DHA intake can identify pregnancies that benefit from high dose DHA supplementation. STUDY DESIGN: This secondary analysis used birth data from 1310 pregnant women who completed a 7-question food frequency questionnaire (DHA-FFQ) at 16.8 ± 2.5 weeks gestation that is validated to assess DHA status. They were then randomly assigned to a standard (200 mg/day) or high dose (800 or 1000 mg/day) DHA supplement for the remainder of pregnancy. Bayesian logistic regressions were fitted for early preterm birth and preterm birth as a function of DHA intake and assigned DHA dose. RESULTS: Participants who consumed less than 150 mg/day DHA prior to 20 weeks' gestation (n = 810/1310, 58.1%) had a lower Bayesian posterior probability (pp) of early preterm birth if they were assigned to high dose DHA supplementation (1.4% vs 3.9%, pp = 0.99). The effect on preterm birth (<37 weeks) was also significant (11.3% vs 14.8%, pp = 0.97). CONCLUSION: The DHA-FFQ can identify pregnancies that will benefit most from high dose DHA supplementation and reduce the risk of preterm birth. The DHA-FFQ is low burden to providers and patients and could be easily implemented in obstetrical practice.

Early and late preterm birth rates in participants adherent to randomly assigned high dose docosahexaenoic acid (DHA) supplementation in pregnancy.

BACKGROUND: Intention-to-treat analyses do not address adherence. Per protocol analyses treat nonadherence as a protocol deviation and assess if the intervention is effective if followed. OBJECTIVE: To determine the rate of early preterm birth (EPTB, <34 weeks gestation) and preterm birth (PTB, <37 weeks gestation) in participants who adhered to a randomly assigned docosahexaenoic acid (DHA) dose of 1000 mg/day. STUDY DESIGN: Eleven hundred women with a singleton pregnancy were enrolled before 20-weeks' gestation, provided a capsule with 200 mg/day DHA and randomly assigned to two additional capsules containing a placebo or 800 mg of DHA. In the Bayesian Adaptive Design, new randomization schedules were determined at prespecified intervals. In each randomization, the group with the most EPTB was assigned fewer participants than the other group. Adherence was defined a priori as a postpartum red blood cell phospholipid DHA (RBC-PL-DHA) ≥5.5%.and post hoc as ≥8.0% RBC-PL-DHA, the latter after examination of postpartum RBC-PL-DHA. Bayesian mixture models were fitted for gestational age and dichotomized for EPTB and PTB as a function of baseline RBC-PL-DHA and dose-adherence. Bayesian hierarchical models were also fitted for EPTB by dose adherence and quartiles of baseline RBC-PL-DHA. RESULTS: Adherence to the high dose using both RBC-PL-DHA cut points resulted in less EPTB compared to 200 mg [Bayesian posterior probability (pp) = 0.93 and 0.92, respectively]. For participants in the two lowest quartiles of baseline DHA status, adherence to the higher dose resulted in lower EPTB (≥5.5% RBC-PL-DHA, quartiles 1 and 2, pp = 0.95 and 0.96; ≥8% RBC-PL-DHA, quartiles 1 and 2, pp = 0.94 and 0.95). Using the Bayesian model, EPTB was reduced by 65%, from 3.45% to 1.2%, using both cut points. Adherence also reduced PTB before 35, 36 and 37 weeks using both cut points (pp ≥ 0.95). In general, performance of the nonadherent subgroup mirrored that of participants assigned to 200 mg. CONCLUSION: Adherence to high dose DHA reduced EPTB and PTB. The largest effect of adherence on reducing EPTB was observed in women with low baseline DHA levels. CLINICALTRIALS: gov (NCT02626299).

Utility of a 7- question online screener for DHA intake.

The secondary analyses of two large, recently completed randomized clinical trials of DHA supplementation in pregnancy found that women with a low baseline DHA status benefited from randomization to a higher dose (800 vs 0 and 1000 vs 200 mg/day DHA). To obtain DHA status, it is necessary to obtain a blood sample and conduct an analysis using gas chromatography (GC) or GC-mass spectrometry (GCMS), both barriers to clinics where pregnant women receive advice on nutrition. Participants consuming less than 150 mg/day of DHA at baseline in our recent trial had a lower risk of early preterm birth and preterm birth when assigned to 1000 vs 200 m/day DHA. DHA intake was determined using a 7-question food frequency questionnaire administered by a trained nutritionist. Because the need for trained personnel to administer the questionnaire would be a barrier to implementing this finding in clinical management of pregnancy, the goal of this study was to determine if an online version of the questionnaire could be validly completed without assistance.

Validation of an abbreviated food frequency questionnaire for estimating DHA intake of pregnant women in the United States.

Docosahexaenoic acid (DHA) intake was estimated in pregnant women between 12- and 20-weeks' gestation using the National Cancer Institute's (NCI) Diet History Questionnaire-II (DHQ-II) and a 7-question screener designed to capture DHA intake (DHA Food Frequency Questionnaire, DHA-FFQ). Results from both methods were compared to red blood cell phospholipid DHA (RBC-DHA) weight percent of total fatty acids. DHA intake from the DHA-FFQ was more highly correlated with RBC-DHA (rs=0.528) than the DHQ-II (rs=0.352). Moreover, the DHA-FFQ allowed us to obtain reliable intake data from 1355 of 1400 participants. The DHQ-II provided reliable intake for only 847 of 1400, because many participants only partially completed it and it was not validated for Hispanic participants. Maternal age, parity, and socioeconomic status (SES) were also significant predictors of RBC-DHA. When included with estimated intake from the DHA-FFQ, the model accounted for 36% of the variation in RBC-DHA.

Higher maternal weight is related to poorer fetal autonomic function.

Maternal obesity is an established risk factor for poor infant neurodevelopmental outcomes; however, the link between maternal weight and fetal development in utero is unknown. We investigated whether maternal obesity negatively influences fetal autonomic nervous system (ANS) development. Fetal heart rate variability (HRV) is an index of the ANS that is associated with neurodevelopmental outcomes in the infant. Maternal-fetal magnetocardiograms were recorded using a fetal biomagnetometer at 36 weeks (n = 46). Fetal HRV was represented by the standard deviation of sinus beat-to-beat intervals (SDNN). Maternal weight was measured at enrollment (12-20 weeks) and 36 weeks. The relationships between fetal HRV and maternal weight at both time points were modeled using adjusted ordinary least squares regression models. Higher maternal weight at enrollment and 36 weeks were associated with lower fetal HRV, an indicator of poorer ANS development. Further study is needed to better understand how maternal obesity influences fetal autonomic development and long-term neurodevelopmental outcomes.

Docosahexaenoic acid supplementation (DHA) and the return on investment for pregnancy outcomes.

The Kansas University DHA Outcomes Study (KUDOS) found a significant reduction in early preterm births with a supplement of 600mg DHA per day compared to placebo. The objective of this analysis was to determine if hospital costs differed between groups. We applied a post-hoc cost analysis of the delivery hospitalization and all hospitalizations in the following year to 197 mother-infant dyads who delivered at Kansas University Hospital. Hospital cost saving of DHA supplementation amounted to $1678 per infant. Even after adjusting for the estimated cost of providing 600mg/d DHA for 26 weeks ($166.48) and a slightly higher maternal care cost ($26) in the DHA group, the net saving per dyad was $1484. Extrapolating this to the nearly 4 million US deliveries per year suggests universal supplementation with 600mg/d during the last 2 trimesters of pregnancy could save the US health care system up to USD 6 billion.

Predicting the effect of maternal docosahexaenoic acid (DHA) supplementation to reduce early preterm birth in Australia and the United States using results of within country randomized controlled trials.

The DHA to Optimize Mother Infant Outcome (DOMInO) and Kansas DHA Outcomes Study (KUDOS) were randomized controlled trials that supplemented mothers with 800 and 600mg DHA/day, respectively, or a placebo during pregnancy. DOMInO was conducted in Australia and KUDOS in the United States. Both trials found an unanticipated and statistically significant reduction in early preterm birth (ePTB; i.e., birth before 34 weeks gestation). However, in each trial, the number of ePTBs were small. We used a novel Bayesian approach to estimate statistically derived low, moderate or high risk for ePTB, and to test for differences between the DHA and placebo groups. In both trials, the model predicted DHA would significantly reduce the expected proportion of deliveries in the high risk group under the trial conditions of the parent studies. Among the next 300,000 births in Australia we estimated that 1112 ePTB (95% credible interval 51-2189) could be avoided by providing DHA. And in the USA we estimated that 106,030 ePTB (95% credible interval 6400 to 175,700) could be avoided with DHA.

Sex-related differences in diurnal activities and development of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7alpha-hydroxylase.

The activities of hepatic microsomal 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase were consistently higher (up to 3-fold) in female compared to male rats fed 2% cholestyramine for 8 h daily. In all animals studied, enzymic activities were highest 6 h after feeding began. However, 85% of the rise in cholesterol 7alpha-hydroxylase activity occurred in the 6 h before and 89% of the rise in 3-hydroxy-3-methylglutaryl CoA reductase activity occurred in the 6 h after feeding started. Sex-related differences in both enzymic activities first became apparent at the time of sexual maturity. Enzymic activities before weaning were generally low and a late-suckling (13--20 days) rise in cholesterol 7alpha-hydroxylase was not accompanied by a rise in 3-hydroxy-3-methylglutaryl CoA reductase. For all of these studies we assayed cholesterol 7alpha-hydroxylase at two concentrations of exogenous cholesterol to obviate problems relating to size of the cholesterol pool.

Evidence that physiologic levels of circulating estrogens and neonatal sex-imprinting modify postpubertal hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

Intact, but sham-operated female rats had 2- to 3-fold higher levels of hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity than their male counterparts (15--21.5 vs. 6.7--8.7 nmol mevalonate/mg protein per h). The activity of the hepatic enzyme declined to about the same relative degree (40--60%) in male and female rats that were gonadectomized after puberty (53 days of age) and killed 5 weeks later. Implantation of silastic capsules containing 17 beta-estradiol increased the level of hepatic 3-hydroxy-3-methylglutaryl CoA reductase to levels found in sham-operated controls. In rats that were gonadectomized in infancy (12 h old) and killed 7--8 weeks later, the level of enzyme activity was not altered in females, but it was increased from 60--240% in males. Consequently, following neonatal gonadectomy, male-female differences in enzyme activity were no longer apparent. Implantation of silastic capsules containing estradiol in neonatally gonadectomized rats resulted in a doubling of enzyme activity in both males and females. Ovariectomy reduced plasma estrogen levels, but implantation of estradiol in gonadectomized males and females increased the hormone level to that found in sham-operated females. Thus, the results strongly suggest a role for physiologic levels of estrogen as a positive effector of 3-hydroxy-3-methylglutaryl CoA reductase activity. Neonatal sex imprinting also appears to modulate the enzyme activity since sex-mediated differences are effaced by gonadectomy in infancy, but not by gonadectomy following puberty.

Docosahexaenoic acid (DHA) supplementation in pregnancy differentially modulates arachidonic acid and DHA status across FADS genotypes in pregnancy.

Some FADS alleles are associated with lower DHA and ARA status assessed by the relative amount of arachidonic acid (ARA) and docosahexaenoic acid (DHA) in plasma and red blood cell (RBC) phospholipids (PL). We determined two FADS single nucleotide polymorphisms (SNPs) in a cohort of pregnant women and examined the relationship of FADS1rs174533 and FADS2rs174575 to DHA and ARA status before and after supplementation with 600mg per day of DHA. The 205 pregnant women studied were randomly assigned to placebo (mixed soy and corn oil) (n=96) or 600mg algal DHA (n=109) in 3 capsules per day for the last two trimesters of pregnancy. Women homozygous for the minor allele of FADS1rs174533 (but not FADS2rs174575) had lower DHA and ARA status at baseline. At delivery, minor allele homozygotes of FADS1rs174533 in the placebo group had lower RBC-DHA compared to major-allele carriers (P=0.031), while in the DHA-supplemented group, all genotypes had higher DHA status compared to baseline (P=0.001) and status did not differ by genotype (P=0.941). Surprisingly, DHA but not the placebo decreased ARA status of minor allele homozygotes of both FADS SNPs but not major allele homozygotes at delivery. Any physiological effects of changing the DHA to ARA ratio by increasing DHA intake appears to be greater in minor allele homozygotes of some FADS SNPs.

Long chain polyunsaturated fatty acid supplementation in infancy increases length- and weight-for-age but not BMI to 6 years when controlling for effects of maternal smoking.

Long chain polyunsaturated fatty acids (LCPUFA) are added to infant formula but their effect on long-term growth of children is under studied. We evaluated the effects of feeding LCPUFA-supplemented formula (n = 54) compared to control formula (n = 15) throughout infancy on growth from birth-6 years. Growth was described using separate models developed with the MIXED procedure of SAS(®) that included maternal smoking history and gender. Compared to children fed control formula, children who consumed LCPUFA supplemented formula had higher length-/stature-/and weight-for-age percentiles but not body mass index (BMI) percentile from birth to 6 years. Maternal smoking predicted lower stature (2-6 years), higher weight-for-length (birth-18 months) and BMI percentile (2-6 years) independent of LCPUFA effects. Gender interacted with the effect of LCPUFA on stature, and the relationship between smoking and BMI, with a larger effect for boys. Energy intake did not explain growth differences. A relatively small control sample is a limitation.

Dual-energy X-ray absorptiometry studies of bone mineral status in newborn infants.

We studied bone mineral status using dual-energy X-ray absorptiometry (DXA) on 150 singleton newborn infants with birth weights 1002-3990 g and gestational ages (GA) 27-42 weeks. Eighty-five infants were preterm (< 38 weeks), and 79 infants were low birth weight (< or = 2500 g). In addition, we aimed to determined the predictive value of anthropometric measurements, race, and gender on variability in bone mineral status. Data were acquired using a whole body DXA scanner with a pediatric platform. Scan analyses were performed with software version V5.64P. Results showed a highly significant (p < 0.001 for all comparisons) correlation among the continuous independent variables, gestational age, birth weight, study weight, study bare weight, and study length, and between independent and each of the dependent variables, total body bone mineral content (TB BMC), TB area, and TB bone mineral density (TB BMD). The best single determinant of bone mineral status is body weight, accounting for 95% of TB BMC and TB area and for 86% of TB BMD variation. Body length was the only additional significant predictor of TB area. Inclusion of postnatal age (during the first week after birth), race, gender, or season, either individually or in combination, failed to improve bone mineral status explanation. By term (GA 38-42 weeks, birth weight 2700-3990 g), the mean TB BMC was 68.2 g, TB area 307.6 cm2, and TB BMD 0.221 g/cm2. We conclude that DXA can be performed even in small preterm infants during the newborn period. Our results can be used as a basis for further studies in physiologic and pathologic situations that might affect bone mineralization in infants.

Behavioral methods used in the study of long-chain polyunsaturated fatty acid nutrition in primate infants.

Domains of behavior may be broadly categorized as sensory, motor, motivational and arousal, cognitive, and social. Differences in these domains occur because of changes in brain structure and function. Docosahexaenoic acid (DHA; 22:6-23) and arachidonic acid (AA; 20:4-26) are major structural components of the brain that decrease when diets deficient in the essential fatty acids (EFA) alpha-linolenic acid and linoleic acid are consumed. Early electrophysiologic and behavioral studies in EFA-deficient rodents showed behavioral effects attributable to lower-than-normal accumulation of DHA and AA in the brain. More recently, electrophysiologic and behavioral studies in EFA-deficient primate infants and analogous studies in human infants have been conducted. The human infants were fed formulas that could result in lower-than-optimal accumulation of long-chain polyunsaturated fatty acids (LCPUFAs) in the brain during critical periods of development. This article describes the behavioral methods that have been used to study primate infants. These methods may be unfamiliar to many physicians and nutritionists who wish to read and interpret the human studies. The behavioral outcomes that have been evaluated in LCPUFA studies represent only a fraction of those available in the behavioral sciences. Specific developmental domains have been studied less often than global development, even though studies of nonhuman primates deficient in EFAs suggest that the former provide more information that could help target the underlying mechanisms of action of LCPUFAs in the brain.

N-acetylneuraminic acid concentrations in human milk oligosaccharides and glycoproteins during lactation.

Human milk contains a variety of N-acetylneuraminic acid (NANA)-containing oligosaccharides, but the expected range of intake of sialic acid in this form by infants fed human milk is unknown. Two quite different amounts have been reported: 120 mg/liter in pooled, mature human milk (1) and 1400 mg/liter in the milk of a single woman on the 1st day of lactation (2). The normal range of NANA intake in human milk glycoproteins likewise does not appear to have been analyzed previously. Data presented here indicate that both human milk oligosaccharide and glycoprotein NANA decline exponentially over the first 2 months of lactation, decreasing little thereafter. During the first 2 months of lactation, milk from women delivering at term cannot be distinguished from that of women delivering significantly before term (less than 32 wks gestation) with regard to oligosaccharide and glycoprotein NANA. The parallel decrease of sialic acid in these fractions suggests a relationship between sialydation of human milk oligosaccharides and glycoproteins. Human milk NANA concentrations are discussed with regard to reports that exogenous administration of NANA can increase cerebral and cerebellar concentrations of NANA in glycoproteins and gangliosides, and produce long term changes in behavior in rats.

Effect of long-chain n-3 fatty acid supplementation on visual acuity and growth of preterm infants with and without bronchopulmonary dysplasia.

Healthy preterm infants fed formula with long-chain n-3 fatty acids (n-3 LCFAs) from marine oil have better early visual acuity but lower plasma phosphatidylcholine (PC) arachidonic acid (AA) and growth than infants fed formula containing linolenic acid (LLA) as the sole n-3 fatty acid. This randomized, double-blind trial was designed to study the effects of a different source of n-3 LCFAs and a shorter feeding interval on visual acuity (by Teller Acuity Card) and growth of preterm infants (n = 59; 747-1275 g birth wt), some of whom required long periods of supplemental oxygen and developed bronchopulmonary dysplasia (BPD). Infants were studied at 0, 2, 4, 6, 9, and 12 mo past term. Plasma PC AA, and normalized weight, length, and head circumference were not influenced by BPD or n-3 LCFAs except that n-3 LCFA-supplemented infants weighed less at 6 (P<0.05) and 9 (P<0.01) mo and had smaller head circumferences at 9 mo (P<0.05). Compared with control infants, however, those fed n-3 LCFAs had lower weight-for-length at 2, 6, 9, and 12 mo (P<0.0003, P<0.0114, P<0.0008, and P<0.006, respectively). n-3 LCFAs improved early (2-mo) but not later acuity among infants without BPD (P<0.02). Regardless of diet, infants with BPD had poorer grating acuity at 2 (P<0.0002) and 4 (P<0.04) mo but not thereafter.

Docosahexaenoic acid status of preterm infants at birth and following feeding with human milk or formula.

The docosahexaenoic acid (DHA) status of preterm infants (less than 32 wk gestation) was measured as the molar percent of DHA in individual red blood cell phospholipids: 1) in cord venous blood immediately following delivery, 2) after infants were receiving greater than 60 kcal X kg X day of energy from oral-gastric feedings, and 3) at a mean of 7 wk later. Infants on full feeding received either preterm human milk or formula. The DHA concentration of all phospholipid classes declined between birth and the time at which enteral feedings constituted the primary source of energy. Subsequent feeding with preterm human milk increased the molar percent of red blood cell phospholipid DHA, while DHA declined further in infants fed formula. Infants fed human milk compared to those fed formula had a significantly higher molar percent of DHA in all red blood cell phospholipids studied.

Effect of vitamin A supplementation of intravenous lipids on early vitamin A intake and status of premature infants.

Preterm infants were randomly assigned to receive routine vitamin A supplementation (Regular A) or additional vitamin A in intravenous lipids (High A). Because infants with bronchopulmonary dysplasia (BPD) have poorer vitamin A status than infants who do not develop BPD, High A and Regular A infants were divided by BPD (no or yes) before determining the effects of treatment on intake and plasma concentration of retinol in the first month. Compared with infants without BPD, those with BPD received less retinol (RE.kg-1.d-1) if assigned to Regular A and more if assigned to High A (BPD by vitamin A interaction, P < 0.002). High A-BPD infants compared with Regular A-BPD infants had significantly higher plasma retinol concentrations in the first month. Retinyl palmitate appears to be an effective adjunct to routine vitamin A administration. Infants most likely to benefit from receiving vitamin A in intravenous lipids are those advanced more slowly to full enteral feeding.

Vitamin A status of preterm infants during infancy.

Plasma retinol and retinol-binding protein (RBP) were measured in 67 enterally fed preterm infants (750-1398 g) at 33 +/- 2 wk postconceptional age (PCA), and at regular intervals during infancy. Retinol and RBP declined by 35 +/- 2 wk PCA and remained low at 38 wk after discharge, with the infants fed a term-infant formula. At 38 +/- 2 wk PCA, 48% (32 of 67) of these infants had plasma retinol concentrations less than 0.35 mumol/L. Mean retinol and RBP rose over the next 7 mo, but large numbers of infants (26 of 59 at 48 wk, 10 of 61 at 57 wk) had hyporetinolemia (0.35-0.67 mumol/L). Plasma RBP leveled off at 57 +/- 2 wk PCA and remained low (less than 0.95 mumol/L) in many infants throughout the first year of life. Lower plasma retinol and RBP concentrations at 33 and 38 wk correlated with longer periods of intravenous nutrition. At 57 and 69 wk, lower retinol and RBP correlated with higher birth order. Suboptimal vitamin A status may occur for many months after preterm infants are discharged from the hospital.

Visual-acuity development in healthy preterm infants: effect of marine-oil supplementation.

Docosahexaenoic acid (DHA; 22:6n-3) is important for normal visual development. We hypothesized that preterm infants fed formulas with marine oil as a source of DHA would have better visual acuity than infants fed formulas without marine oil, as measured by the Teller Acuity Card procedure. Marine oil (P < 0.001) and age (P < 0.0001) influenced visual acuity, by repeated-measures analysis of variance (ANOVA) corrected for the effect of subject. Marine-oil-supplemented infants had better visual acuity than those fed standard formulas at 2 and 4 mo of age, by Fishers' least-squares difference (LSD). Acuity of both dietary groups improved through 6.5 mo of age, then plateaued. Through 4 mo of age, acuity was inversely related to oxygen supplementation (log10 h) and positively related to DHA status, by general-linear-models (GLM) analysis. After 4 mo of age, birth weight and gestational age were the only variables consistently related to visual acuity by GLM. We conclude that marine-oil-supplemented formula improved visual acuity of preterm infants through 4 mo of age by improving DHA status.

trans Fatty acids: infant and fetal development.

This review evaluates scientific data associated with the possibility that trans fatty acids compromise fetal and infant early development. Concerns have been triggered by research that has heightened our awareness of the importance of n-3 and n-6 fatty acids; shown that trans fatty acids inhibit delta6 desaturation of linoleic acid; identified trans fatty acid isomers in fetal, infant, and maternal tissues; and reported an inverse association between the trans fatty acid content of tissue lipids and measures of growth and development. Animal studies provide little evidence that trans fatty acids influence growth, reproduction, or gross aspects of fetal development. However, these models may not have been appropriate for addressing all the subtle effects that influence development of human infant retinal, neural, or brain function. Human studies are hampered by the complexity of the interrelations among nutritional, genetic, and environmental factors and by ethical considerations that constrain the research design. Existing data have not established a causal relation between trans fatty acid intake and early development. Conclusions cannot be drawn from the possible association found between trans fatty acid exposure and lower n-3 and n-6 long-chain polyunsaturated fatty acids and growth because of confounding factors. Few studies addressed the question of whether trans fatty acids adversely affect human fetal growth. One study reported a correlation between the trans fatty acid content of plasma and birth weight of preterm infants and one study reported a relation between preterm births and the trans fatty acid content of maternal plasma. Limited associative data have addressed whether trans fatty acids adversely affect fetal and infant neurodevelopment and growth. The interpretation of existing research and development of recommendations should be done cautiously. Suggestions for research to clarify these issues are made.