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2

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Biometry and Nutrition Unit, Agharkar Research Institute, Pune, India;
King Edward Memorial Hospital Research Center, King Edward Memorial Hospital, Pune, India;
**
Medical Research Council Environmental Epidemiology Unit, Southampton, U.K. and
Institute of Human Nutrition, University of Southampton, Southampton, U.K.
2To whom correspondence should be addressed at Diabetes Unit, King Edward Memorial Hospital, Rasta Peth, Pune, Maharashtra, India 411 011. E-mail: diabetes{at}vsnl.com
| ABSTRACT |
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KEY WORDS: India maternal intake food frequency questionnaire green leafy vegetables birth size
| INTRODUCTION |
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Studies that investigated the relationship between maternal nutrition
and babies birth size are scarce, and those available are inconsistent
(Susser 1991
). This relationship is influenced by many
biological and socioeconomic factors, which vary widely in different
populations. For example, the relationship differs among adolescents
(Scholl et al. 1994
), among women from a low
socioeconomic class (Hediger et al. 1994
) and even in
most developed countries like Austria, where women have cosmetic
undernutrition (Kirchengast and Hartmann 1998
). Studies
of energy and protein supplementation during pregnancy have produced
varying and sometimes conflicting results (Kramer 1993
),
although there is some evidence that supplementation may be beneficial
in very marginally nourished women (Ceesay et al. 1997
).
The dietary intakes of energy and protein of rural Indian mothers are
low (Bhatia et al. 1981
, Grover 1982
,
Hutter 1996
, Piers et al. 1995
,
Rawtani and Varma 1989
, Vijayalaxmi and Lakshmi 1985
, Vijayalaxmi et al. 1988
). The consumption
of foods that are important sources of micronutrients, such as dairy
products, meat, fresh fruits and green leafy vegetables
(GLV),3
is also low in rural Indian populations (Gupta and Sharma 1980
). Rural Indian women are often engaged in a high level of
physical activity (Piers et al. 1995
). The majority of
previous studies in India have examined maternal diets in terms of
quantity (macronutrients) using 24-h recall method but rarely assessed
quality (micronutrients). Food frequency questionnaires (FFQ) that are
likely to offer estimates of habitual intake have rarely been used in
studies of pregnant women.
The assessment of maternal nutritional status requires the measurement
of body composition (before and during pregnancy), determination of
energy intake and workload, measurement of patterns of food intake,
consumption of macronutrients and micronutrients and biochemical
measurements of micronutrient status. In the Pune Maternal Nutrition
Study, we set out to relate the nutritional status before and during
pregnancy of women living in rural Maharashtra, India, to the birth
weight and other measurements of their babies. We reported earlier
(Fall et al. 1999
) that the size at birth is strongly
predicted by maternal prepregnancy weight and weight gain in pregnancy.
Maternal height, head circumference and prepregnancy fat mass
independently predicted the birth weight of the baby. The present
report describes our findings in relation to maternal macronutrient
intakes, intakes of micronutrient-rich foods and biochemical
micronutrient status. For biochemical micronutrient status, we focused
on folate, iron and vitamin C. Folate and iron have traditionally been
considered important micronutrients for fetal growth, and vitamin C is
crucial for iron absorption.
| MATERIALS AND METHODS |
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The study took place in six villages, 4050 km from Pune City,
and covered a population of
35,000. Of 2675 married eligible women
(aged 1540 y), 2466 women (92%) agreed to participate. Field workers
visited them every month to record the date of their last menstrual
period; women who missed two successive periods were examined with
ultrasound at 1518 wk to record sonographic gestational age
(Hadlock 1990
). Gestational age was derived from the
last menstrual period, unless it differed from the sonographic estimate
by >2 wk, in which case the latter estimate was used. Women entered
the study if a singleton pregnancy of <21 wk gestation was confirmed.
Socioeconomic status was assessed using a standardized questionnaire
(Pareek and Trivedi 1964
), which derives a composite
score based on occupation and education of the head of the household,
caste, type of housing and family ownership of animals, land and
material possessions. The majority of women were poorly educated and
belonged to subsistence farming families. All women were given 100
tablets of iron (60 mg) and folic acid (0.5 mg) at 18 wk gestation
according to the National Nutritional Anemia Control Program.
During the study period, June 1994 through April 1996, 1102 women reported missing periods. One hundred twelve women reported an abortion/termination, 8 had major fetal anomalies and 3 had multiple pregnancies. Fourteen had incomplete prepregnancy anthropometry, and 168 were entered beyond 21 wk of gestation. Thus, 797 women were studied for this analysis. Ethical permission for the study was granted by the King Edward Memorial Hospital Ethical Committee and by the local village leaders.
Nutritional assessment
Anthropometric measurements. Women were measured every 3 mo to record their weight, height, skinfold at four sites and head and mid-upper arm circumference (MUAC). The last set of measurements made before pregnancy was used as prepregnancy anthropometry, and the measurements were repeated during pregnancy at 18 ± 2 and 28 ± 2 wk gestation.
Dietary intake.
The conventional 24-h recall method was modified and made more
objective by incorporating information on portion sizes, which were
weighed at each mealtime by a trained fieldworker. Women were
interviewed at 18 and 28 wk of gestation by one of four nutritionists
to record the consumption of food items in chronological order from
morning until dinnertime. At the time of diet survey, interviewers
ensured that the woman was not fasting or had an illness and that she
reported foods consumed outside the home on the day of the visit. A
database of nutritive values of foods was generated by analyzing 288
distinct food preparations commonly consumed in the community. Protein
was estimated in dried samples by the micro-Kjeldhal method, using
the 1030 Kjeltec Autoanalyser system (Tecator AB, Hoganas,
Sweden). Fat was estimated using the Soxlet method, in which food
samples are subjected to continuous ether extraction for 18 h
(Raghuramulu et al. 1983
). Carbohydrates were estimated
by subtraction. In addition to the 24-h recall questionnaire, a FFQ was
administered to obtain frequency of consumption of 111 foods in 17 food
categories during the preceding 3-mo period, on an 8-point scale
ranging from "never" to "more than once daily." The 17 food
categories were beverages, chapati/roti, rice, pulses, legumes,
vegetables, GLV, chutneys, fasting foods, fruits, meat/fish, milk
products, bakery items, spicy snacks, sweet snacks, festival foods and
special foods. The food groups were mutually exclusive.
Biochemical measures of maternal nutritional status.
In addition to dietary intakes, maternal red cell folate and serum
ferritin and vitamin C concentrations were measured at 18 and 28 wk.
Blood samples were taken from fasting subjects at an early-morning
home visit. Samples were protected from light and transferred within
1 h via motorcycle to a local village research center, where
samples were centrifuged at 1310 x g, and the
serum was separated. Samples were transferred, at 04°C in a
light-protected box, to the main laboratory at the King Edward
Memorial Hospital in Pune. EDTA samples for red cell folate assay were
diluted (1:19 by volume) with 10 g ascorbic acid/L solution and
incubated for 90 min at room temperature, before being frozen at
-80°C. An equal volume of 100 g metaphosphoric acid/L was added
to 0.5-mL serum aliquots for vitamin C assay, before being frozen at
-80°C. Samples were transferred to the United Kingdom on dry ice.
Red cell folate and serum ferritin concentrations were measured in the
Hematology Laboratory, Southampton General Hospital (Southampton, U.K.)
with radioimmunoassays (Becton Dickinson U.K., Oxford, U.K.). Serum
vitamin C concentrations were measured at the Medical Research Council
Human Nutrition Research Center (Cambridge, U.K.) using an ascorbate
oxidaseorthophenylene diamine assay on a Roche Cobas Bio Centrifugal
analyzer (Hoffman-LaRoche, Basel, Switzerland) with a fluorescence
attachment (Vuilleumier and Keck 1989
).
Physical workload assessment
The womens typical daily physical activity was recorded at 18
and 28 wk gestation using simple numeric measures in a specially
designed activity questionnaire, which included farming and domestic
activities. For example, domestic activities, such as cooking and
washing clothes/utensils, were recorded in terms of the number of
people catered for, whereas fetching water was recorded in terms of the
number of trips and number of containers carried. Using published data
on the energy cost of various activities (Bleiberg et al. 1981 and 1980
, Lawrence et al. 1985
), a weighted
total daily score was derived. This score reflected as a base unit, an
activity level of 1 kcal (4.184 kJ)/min for a 30-min slot of time. The
questionnaire was validated in 41 women using a 1-d minute-to-minute
observer maintained records. Activity scores obtained by questionnaire
were significantly correlated (P < 0.05) with actual
time spent in domestic (r = 0.34) and farming
(r = 0.56) activities. The total scores were used to
rank women into tertiles of "low," "medium" and "high"
physical activity.
Neonatal anthropometry
Babies were measured by one of five trained fieldworkers within 72 h of birth. Birth weight was measured to the nearest 50 g using a Salter spring balance (Salter Abbey, Suffolk, U.K.); crown-heel length was measured to the nearest 0.1 cm using a portable Pedobaby Babymeter (ETS J.M.B., Brussels, Belgium). Triceps and subscapular skinfold thicknesses were measured to the nearest 0.2 mm, on the left side of the body, using Harpenden skinfold calipers (CMS Instruments, London, U.K.). Occipitofrontal head circumference and MUAC were measured to the nearest 0.1 cm using Fiberglas tapes (CMS Instruments, London, U.K.). Abdominal circumference was measured at the level of the umbilicus in expiration. Placental weight was recorded to the nearest 5 g using Ishida scales after trimming of the umbilical cord and membranes. Interobserver and intraobserver variation studies were conducted every 3 mo to ensure quality of these measurements.
Statistical methods
Differences between group means were tested using t tests. Multiple regression analysis was used to examine trends in birth size according to maternal dietary intakes and biochemical data and to assess the relative contributions of other factors to the variability in birth measurements. In regressions, birth measurements, maternal measurements, energy and macronutrient intakes and erythrocyte folate, vitamin C and ferritin concentrations were analyzed as continuous variables. Intakes of specific foods based on the FFQ and socioeconomic and activity scores were analyzed as grouped variables. However, when using the regression analysis, we weighted these groupings to reflect as closely as possible the frequency of consumption per week. All analyses were adjusted for the babys sex, gestational age at delivery and maternal parity. Maternal and neonatal skinfold measurements were skewed and required log-transformation to satisfy assumptions of normality. Analysis was carried out using SPSS/PC+, Version 5.0. Values unless otherwise stated are means ± SD.
| RESULTS |
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Mothers
The mean age of the mothers was 21.4 y, and 31.6% were
primiparous. They were short, light and thin (Table 1
). Twenty-three percent of the women had a prepregnancy body
weight of <38 kg, and 9% were shorter than 145 cm, considered high
risk for low birth weight (Gopalan 1989
). Of the women,
31.3% had a body mass index (BMI) of <17 kg/m2,
indicating severe chronic energy deficiency (World Health Organization 1995
). Weight gain during pregnancy was 2.1
± 2.8 kg up to 18 wk and 5.5 ± 2.9 kg up to 28 wk. All
measurements except maternal MUAC showed a significant increase up to
28 wk gestation.
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The full-term birth weight was 2665 g (Table 1)
, and 28%
of babies were of low birth weight (<2500 g). Even among full-term
babies, birth weight increased with gestational age (r
= 0.36). Birth weight, length and head circumference were greater
in boys than in girls (P < 0.01). All measurements
except length, head circumference and MUAC were smaller in babies born
to primiparous than in babies born to multiparous mothers (P
< 0.05). In our analysis of birth size in relation to nutritional
data, we therefore adjusted for gestational age at delivery, babys
sex and maternal parity.
Dietary intakes
Macronutrients (24-h recall).
Maternal energy and protein intakes at 18 and 28 wk were energy of 7.4
± 2.1 and 7.0 ± 2.0 MJ and protein of 45.4 ± 14.1 and
43.5 ± 13.5 g, respectively (Table 2
). These values are low compared with Recommended Daily Allowances for
Indian pregnant women given by the Indian Council of Medical Research (1987
). Carbohydrates were the main energy source
(72%), whereas 10 and 18% of energy was derived from protein and fat,
respectively. Most protein was derived from cereals and pulses. Only
38% of women consumed animal protein, which contributed only 15% to
the daily protein intake. Activity scores at 18 wk gestation were high
(82.3 ± 21.0), especially among women from farming families, and
remained so at 28 wk gestation (76.6 ± 23.2).
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Food groups (FFQ). Of the 17 food groups assessed using the FFQ, significant relationships with birth size were found with GLV, fruits and milk products.
GLV.
The GLV eaten frequently (more than once a week) in this community were
fenugreek leaves (57% of women), spinach (33%), coriander (16%) and
colocasia (15%). The frequency of consumption of GLV at 28 wk was
strongly related to all birth measurements (Table 3
). These relationships remained significant after adjustment for
prepregnancy weight (or height and BMI), energy intakes, physical
activity score, weight gain during pregnancy and socioeconomic status
(Table 4
). An increase in frequency of consumption from one group to the next
higher group was associated with an increase in birth weight of 19 g [95% confidence interval (CI), 830] after adjustments for all of
these factors. The trend with birth weight was stronger (value of
partial regression coefficient increased to 30 g; 95% CI, 1347)
among the lightest mothers, those with a prepregnancy weight below the
lowest tertile (40 kg). The odds ratio for delivering a low birth
weight baby was 0.43 (95% CI, -0.12 to 0.99) in mothers who ate GLV
at least every other day compared with 1.0 in mothers who never ate
them.
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Milk products.
Milk was consumed mostly by women from families who owned
milk-producing animals. It was consumed either with roti or rice or
was drunk but rarely was consumed in the form of other milk products.
Milk consumption was strongly associated with socioeconomic score
(P < 0.001). The frequency of milk consumption at 18
wk gestation was related to birth weight, birth length, MUAC, head
circumference and placental weight but not to measures of neonatal fat
or abdominal circumference (Table 3)
. In contrast to GLV and fruits,
these relationships were stronger at 18 wk than at 28 wk gestation.
They were similar at all levels of prepregnancy maternal weight and
remained significant after adjustment for prepregnancy weight (or
height and BMI), energy intakes, physical activity score, socioeconomic
status and weight gain (Table 4)
.
In all of these regression analyses, the major contribution was from
main confounding variables (gestation, sex and parity:
20%). The
contributions of dietary variables varied from 2 to 3% but were
significant.
Biochemical indices of maternal nutrition
Concentrations of erythrocyte folate, serum ferritin and serum
vitamin C [median (interquartile range)] were 868 nmol/L (6871097),
13 µg/L (823) and 10 µmol/L (231) at 18 wk gestation and 968
nmol/L (7411273), 10 µg/L (720) and 6 µmol/L (122) at 28 wk
gestation. All three micronutrients were related to birth size
(Table 5
). The strongest and most consistent relationship was with 28-wk
erythrocyte folate concentration, which was positively related to birth
weight, birth length, head circumference, MUAC, abdominal circumference
and placental weight but not to skinfold thickness. Higher serum
vitamin C concentration at 28 wk gestation was associated with higher
birth length, MUAC and abdominal circumference (Table 5)
, and higher
concentration at 18 wk gestation was associated with higher MUAC
(P < 0.05) and triceps skinfold thickness
(P < 0.05). In contrast, ferritin concentration at 28
wk gestation was inversely related to birth length, MUAC and abdominal
circumference (Table 5)
. All of these relationships remained
significant after adjustment for prepregnancy weight (or height and
BMI), energy intakes, physical activity, weight gain and socioeconomic
status.
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| DISCUSSION |
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Rural mothers were thin and short, indicating that many were
chronically energy deficient before conception. Although their mean
weights and heights were similar to those reported from other rural
areas of India (Vijayalaxmi et al. 1988
), they were
significantly less than those of urban affluent Indian (Devi et al. 1989
, Gupta and Sharma 1980
, Piers et al. 1995
) and Western (Godfrey et al. 1996
)
mothers. The neonatal size was similar to that reported in other Indian
populations (Mohan et al. 1990
) but markedly smaller
than that of Western babies (Godfrey et al. 1996
).
Maternal intakes of energy and protein were low,
7075% of
recommended intakes (ICMR 1987
) at both time points, and
showed no significant relationships with neonatal size. For energy
intakes, the lack of any relationship with birth size was true for the
whole study group, within subgroups of maternal prepregnancy weight and
after taking physical activity into account. It was also true when
women with extremely low intakes (<1.2 basal metabolic rate,
n = 150) were excluded. These important negative
findings are consistent with the slight effects on birth size of energy
and protein supplementation trials in pregnancy (Kramer 1993
).
Among the macronutrients, only fat intake at 18 wk gestation showed an
association with birth size. Fat intake has been shown to correlate
with birth weight (Doyle et al. 1982
), but there are few
data for fat reported as an entity separate from energy. Recent studies
suggest that specific fatty acids are important for fetal growth
(Crawford et al. 1989
), which brings into question
whether fat is a macronutrient or micronutrient.
Birth size was strongly related to intakes of GLV and fruits at 28 wk gestation and of milk at 18 wk gestation. These three food groups are particularly rich in micronutrients. Our observations therefore suggest the importance of specific micronutrients, or their combinations, for fetal growth. For example, GLV are a rich source of folate, iron, provitamin A carotenoids and antioxidants. Increased frequency of the consumption of GLV was associated with an increase in all neonatal anthropometry, and the relationship with birth size remained significant even after correction for red cell folate concentration in blood, suggesting that nutrients other than folate contribute to the relationship. Similarly, fruits are rich in vitamin C and other antioxidants, whereas milk provides high quality proteins, fat, calcium, riboflavin and vitamins A and D. It was interesting to note that a greater consumption of fruits or milk was not associated with increased measures of neonatal fat or abdominal circumference, as occurred for the consumption of GLV. Furthermore, the relationship with fruit consumption when corrected for circulating vitamin C concentration in blood did not remain significant for group as a whole but was significant only for thin mothers. Similarly, the relationship of milk consumption after adjustment for fat intake did not remain significant for birth weight but was significant for length and triceps skinfold thickness. This suggests important roles of different micronutrients in improving fetal growth.
One of the causal pathways for the effect of micronutrients could be an
increase in the gestational period. The exclusion of gestation from
regression analyses in Table 4
showed marginal increase in the
contribution of respective dietary variables in
R2 and keeps the relationship
significant. However, its inclusion explains up to 15% of the
variability in birth weight and substantially increases the overall
R2. This suggests that the effect of
micronutrient intakes on fetal growth is in part mediated through the
lengthening of the gestation.
Birth measurements were related to intakes of GLV and fruit at 28 wk,
whereas they were related to fat and milk intakes at 18 wk. This may
reflect different nutrient requirements for fetal growth at these times
due to the development of different tissues at various stages of
gestation (Dugdale and Payne 1975
, Tanner 1989
). The relationships of GLV and fruit intake to birth size
were strongest in lighter and thinner women. We did not detect any
significant difference in other factors (parity, macronutrient intake,
blood pressure and glycemic status) associated with fetal growth in
these women compared with the remainder of the group. It is likely that
micronutrients may be the most important limiting nutrients in
undernourished women.
We found inverse relationships between maternal serum ferritin
concentration at 28 wk gestation and neonatal size. This somewhat
paradoxical finding has been reported in other populations
(Goldenberg et al. 1996
) and might be explained by
hemodilution effects due to plasma volume expansion, which is
associated with increased fetal growth (Rosso et al. 1992
). Because ferritin represents a stored form of iron, an
alternative explanation is that a lower ferritin concentration in the
mother signifies the successful mobilization of iron, making it
available for fetal growth. It is interesting, in this respect, that
the inverse relationships of serum ferritin concentration were
strongest with neonatal MUAC (representing muscle) and abdominal
circumference (representing viscera), tissues with a high iron content.
To our knowledge, this study is the first to show an association between maternal intakes of GLV, fruits and milk and size at birth . Furthermore, the significant relationship between biomarkers (erythrocyte folate and serum vitamin C) and the frequency of consumption of GLV and fruits, respectively, provides a measure of confidence in our FFQ assessment. Although we cannot assume cause-and-effect relationships in any of the associations we have described, our data suggest that improved maternal intakes of milk in early gestation and of GLV and fruit in late gestation could lead to improved fetal growth. There may be concern in attributing significance to these foods because we had conducted multiple analyses of 17 food groups. We did, however, start with the a priori hypothesis that micronutrient-rich foods may be important for fetal growth. If the P-value was corrected for these 17 food groups, making P = 0.003 the cutoff for statistical significance, the relationship between birth size and GLV remains significant.
There is controversy as to whether food or pharmacology is the best
means of providing micronutrients (West 1996
). There is
some evidence that supplementation with folate in pregnancy leads to
improved fetal growth (Baumslag et al. 1970
, Ek 1982
, Goldenberg et al. 1992
, Iyengar and Rajalakshmi 1975
). However, an evaluation of Indias
long-standing anemia prophylaxis program, with routine iron and
folate supplementation to women in the third trimester of pregnancy for
the past two decades, demonstrates no significant impact on birth
weight (ICMR 1989
). It may be that the
micronutrient-rich foods discussed here provide a more effective
combination of nutrients than do conventional supplements that contain
only one or two micronutrients or macronutrients. Thus, food-based
interventions may be more beneficial. There are limited data from
controlled trials on the effects of micronutrients on fetal growth.
Most have shown a small or no effect (Mathews 1996
,
Onis et al. 1998
). The long-term implications of our
findings that specific maternal food intakes are related to different
components of neonatal phenotype are unknown at this stage and could be
best understood by following these babies to determine infant mortality
rates, childhood growth rates and long-term health. Further
research, in animals and humans, is needed to identify the important
nutrients provided by these key food groups and their mode of action.
We also believe an intervention trial should be attempted to study the
effects of increased availability of these foods for pregnant women.
| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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3 Abbreviations used: BMI, body mass index; FFQ, food frequency questionnaire; GLV, green leafy vegetables; ICMR, Indian Council of Medical Research; MUAC, mid-upper arm circumference. ![]()
Manuscript received April 5, 2000. Initial review completed July 28, 2000. Revision accepted January 2, 2001.
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