Vitamin C supplementation to prevent premature rupture of the

chorioamniotic membranes: a randomized trial

13
Esther Casanueva, Carmina Ripoll, Maricruz Tolentino, Rosa Maria Morales, Frania Pfeffer, Pablo Vilchis, and
Felipe Vadillo-Ortega
ABSTRACT
Background: Vitamin C is involved in the synthesis and degrada-
tion of collagen and is important for maintenance of the chorioam-
niotic membranes. Inadequate availability of ascorbic acid during
pregnancy has been proposed as a risk factor for premature rupture
of the chorioamniotic membranes (PROM).
Objective: The objective of the study was to evaluate the effective-
ness of 100 mg vitamin C/d in preventing PROM.
Design: A controlled double-blind trial was performed. Pregnant
women (n 126) in their 20th wk of gestation were invited; 120
accepted and were randomly assigned to 2 groups (100 mg vitamin
C/d or placebo). Every 4 wk, plasma and leukocyte vitamin C con-
centrations were measured, and each subject was evaluated for cer-
vicovaginal infection. The incidence of PROM was recorded for
each group as an indicator of the protective effect of vitamin C
supplementation.
Results: One hundred nine patients finished the study. Mean plasma
vitamin C concentrations decreased significantly throughout the
pregnancy in both groups (P0.001), and there were no significant
differences between groups. Between weeks 20 and 36, mean leu-
kocyte vitamin C concentrations decreased from 17.5 to 15.23 g/
10
8
cells in the placebo group and increased from 17.26 to 22.17
g/10
8
cells in the supplemented group (within- and between-group
differences: P 0.001). The incidence of PROM was 14 per 57
pregnancies (24.5%) in the placebo group and 4 per 52 pregnancies
(7.69%) in the supplemented group (relative risk: 0.26; 95% CI:
0.078, 0.837).
Conclusion: Daily supplementation with 100 mg vitamin Cafter 20
wk of gestation effectively lessens the incidence of PROM. Am
J Clin Nutr 2005;81:85963.
KEY WORDS Vitamin C, ascorbic acid, pregnancy, prema-
ture rupture of the chorioamniotic membranes, preterm labor, di-
etary reference intakes, DRIs, Mexico
INTRODUCTION
Premature rupture of the chorioamniotic membranes (PROM)
is a complication affecting 1020%of all pregnancies. PROMis
the main known cause of preterm delivery and is associated
worldwide with increased rates of neonatal and maternal mor-
bidity and mortality (1). Thus, it is important to develop public
health strategies to prevent PROM. It has been proposed that
maintaining adequate vitamin C status, ascertained on the basis
of the vitamin Cconcentrations in leukocytes, could help reduce
the incidence of PROM(2, 3). Ina previous report, we (3) showed
that PROMcould be used as a functional test of vitamin Cstatus
throughout pregnancy. Our findings derived from the role of
vitamin C in collagen metabolism and that of collagen in main-
taining the mechanical strength of the chorioamniotic mem-
branes throughout gestation (4, 5). Vitamin C is recognized as a
cofactor for collagenposttranscriptional modifications (6) andas
a down-regulator of the gene transcription of the 72-kDa type IV
collagenase, matrix metalloproteinase (MMP-2), in amnion cells
(7); it also controls the catabolism of collagen.
The current recommended dietary allowance for vitamin C
does not adequately take into account the metabolic adjustments
that occur during pregnancy. Altered patterns of collagen syn-
thesis and diminished concentrations of vitamin C at week 28 of
gestation have been associated with subsequent occurrence of
PROM (3, 4). It has been postulated that low concentrations
of plasma and leukocyte ascorbic acid may result froma dilution
effect caused by hemodynamic adjustments of blood volume
during the third trimester of pregnancy (3). Leukocytes may act
as a store of vitamin C and can provide a more reliable indicator
of vitamin C nutritional status (8) than can the ascorbic acid
concentration in plasma or serum, which more specifically re-
flects the recent consumption of vitamin C. We recently con-
ducted a clinical trial measuring the saturation dose of vitamin C
in pregnant Mexican women (9) and found that 100 mg vitamin
C/d added to the usual diet is sufficient to maintain a leukocyte
ascorbate concentration 18 g (102 nmol)/10
8
cells, and this
concentration protects against PROM (3).
Other conditions commonlypresent duringpregnancy, suchas
cervicovaginal or intrauterine infections (which are themselves
risk factors for PROM), may also affect the nutritional status of
vitamin C (10). Induction of the inflammatory response, which
involves the respiratory burst in leukocytes, causes the consump-
tion of antioxidants such as vitamin C (11). The incidence of
1
Fromthe Public Health Research Branch (EC, CR, MT, RMM, and FP),
the Department of Obstetrics and Gynecology (PV), and the Direction of
Research (FV-O), National Institute of Perinatology, Mexico City, Mexico.
2
Supported by grant no. 783-P from the Consejo Nacional de Ciencia y
Tecnologia, Mexico.
3
Address reprint requests to E Casanueva, Public Health Research
Branch, Instituto Nacional de Perinatologia, Torre de Investigacion, 2o Piso,
Montes Urales 800, Lomas de Virreyes, DF 11000 Mexico. E-mail:
casanuev@servidor.unam.mx.
Received June 9, 2004.
Accepted for publication December 6, 2004.
859 Am J Clin Nutr 2005;81:85963. Printed in USA. 2005 American Society for Clinical Nutrition

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PROM may also reflect the potentiation of chronic or iterative
infections or their synergistic effects during pregnancy and the
presence of low stores of vitamin C (12). The aim of this study
was to evaluate the effectiveness of 100 mg vitamin C/d in pre-
venting PROM.
SUBJECTS AND METHODS
This double-blind study was performed fromSeptember 2002
to August 2003 at the Instituto Nacional de Perinatologia (INPer)
in Mexico City. One hundred twenty-six women attending the
prenatal clinic were identified as eligible participants. Inclusion
criteria were defined as follows: pregnant women with no acute
or chronic diseases, 20 wk of gestation, singleton pregnancy,
no consumption of vitamin supplements, and provision of writ-
teninformedconsent. Exclusioncriteria were the needfor uterine
cerclage or the presence of an obstetric indication for cesarean
delivery.
Women were allocated to 1 of 2 groups by a random-number
table; one group received 100 mg vitamin C/d, and the other
group received a placebo of the same size and shape (both
provided by Roche Pharmaceuticals, Mexico City). The prin-
cipal investigator generated the random table, prepared all
tablets, and ensured that staff members were blinded as to the
grouping codes. Vitamin C or placebo was shipped to each
participant directly from the research pharmacy, which con-
cealed the treatment assignment from the investigators. The
women were instructed to ingest one tablet with water before
breakfast each day.
The sample size was calculated from the overall incidence of
PROM at the INPer (18.5%), regardless of gestational age, and
on the basis of our hypothesis that intervention with vitamin C
wouldreduce the incidence of PROMby20%. This wouldensure
an overall type I error of 5%and an 80%power-to-test reduction
in the incidence of PROM. According to these estimations and in
the anticipation of losses of 10% during the study, we needed to
study 60 cases per group.
Written informed consent was obtained from all subjects. If
the pregnant woman was 18 y old, the written informed con-
sent of a parent or legal guardian was obtained. The protocol was
reviewed and approved by the Research and Ethics Board of the
institution (protocol registration no. 21225049311).
Study protocol
During the study, women were evaluated according to stan-
dardizedprocedures bytrainednutritionists andfacultymembers
of the Obstetrics and Gynecology Department. At the first eval-
uation, each womans general data, gynecologic and obstetric
history, and anthropometric data, including self-reported
prepregnancy weight, were recorded. Information on socioeco-
nomic status according to the scale of the Asociacion Mexicana
de Agencias de Estudios de Mercado (Mexican Association of
Marketing and Opinion Research) was also included; this scale
measures purchasing power and contains 6 categories ranging
from the highest to the lowest purchasing power (13). Each
subject completed a food-frequency questionnaire, which con-
sidered the habitual intake of the previous year and which was
previously validated in Mexican women (14). To obtain infor-
mation on the habitual intake of vitamin C, intakes were cor-
rected for cooking losses by a factor of 50% for boiled or fried
food and by a factor of 25% for steamed food (15).
From weeks 20 to 36, women were evaluated every 4 wk. At
each evaluation, a vaginal exploratory examination was per-
formed, and a vaginal swab was taken for Gram staining and
microbiologic study. The microorganisms of interest included
Gardnerella vaginalis, Candida albicans, Group B streptococ-
cus, Escherichia coli, Neisseria gonorrhoeae, and Ureaplasma
urealyticum. Cervicovaginal infection was diagnosed by clinical
manifestations and positive results of the Gramstain and culture.
If infection was diagnosed, the woman received treatment with
antibiotics in accordance with established clinical protocols.
Asymptomatic subjects with a positive culture did not receive
treatment (16). To evaluate the adherence to treatment, the
women were instructed to maintain a personal record to register
the daily consumption of tablets; the remaining tablets were
counted by research staff at each visit.
At the end of pregnancy, the incidence of PROM was regis-
tered as the primary outcome. PROMwas defined in accordance
withthe AmericanCollege of Obstetricians andGynecologists as
the leakage of amniotic fluid through ruptured chorioamniotic
membranes that occurs 2 h before the onset of labor and after
week 20 of pregnancy (17). The diagnosis of leakage of amniotic
fluid was established clinically by physical examination that
identified the leakage of amniotic fluid through the cervix and
confirmed by crystallization and the nitrazine test. A clinical
evaluation was performed at delivery by using the method of
Capurro et al (18) to verify the gestational age as estimated from
the last menstrual period reported by the subject. Adifference of
1 wk between the 2 evaluations was accepted; when the dif-
ference was 1 wk, the case was excluded from the study.
Pretermlabor was established when the delivery occurred before
37 wk of gestation.
Laboratory tests
During the evaluation visits, a 15-mL sample of blood was
collected into EDTA-coated tubes to measure plasma and leu-
kocyte vitamin C concentrations. On the day of blood sampling,
the women had fasted for 1012 h and had not consumed the
supplement or placebo tablet. Within 30 min after obtaining the
blood sample, the plasma was separated by centrifugation at
1800 g for 10 min, and leukocytes were isolated by using
Dextran T500 (Sigma, St Louis, MO) (19). Leukocytes were
counted with an automatic cell counter (Coulter Counter T890,
version 3D; Beckman Coulter, Miami, FL). After the addition of
methaphosphoric acid (Sigma) to prevent vitamin C oxidation,
the plasma and leukocyte pellets were frozen at 70 C. All
samples were assayed in duplicate by using HPLC with an elec-
trochemical detector (Perkin Elmer Life and Analytic Sciences,
Shelton, CT; 20). Interassay and intraassay CVs were 5% for
all procedures. Laboratory personnel were blind as to patient
group allocation.
Data analysis
We used a repeated-measures analysis of variance with or-
thogonal polynomial contrasts, adjusted for multiple compari-
sons, in the general linear models (GLM) program on SPSS
software (version 11.0; SPSS Inc, Chicago IL) with Tukeys
correction to evaluate changes in vitamin C concentration in
plasma and leukocytes at the return visits. Comparison of the
maternal characteristics, infections, and the incidence of PROM
in the 2 groups was performed by chi-square test. Relative risks
(RRs) with 95% CIs were calculated.
860 CASANUEVA ET AL

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RESULTS
Of 126 participating women, 109 (91%) finished the study: 52
women in the vitamin Csupplemented group and 57 in the
placebo group (Figure 1). The groups did not differ in any
baseline variable suchas age, number of gestations, or nutritional
status. None of the women reported smoking cigarettes during
pregnancy. According to the socioeconomic status scale, the
participants belonged to the lower middle-class, and the 2 groups
did not differ significantly in socioeconomic status. A history of
PROM was reported by 10.6%. The general and outcome vari-
ables of the 2 groups are presented in Table 1.
A cervicovaginal infection at the first evaluation at 20 wk of
pregnancy was present in 32 (29%) of the 109 patients; 14 (25%)
of the 57 women in the placebo group and 18 (35%) of the 52
women in the vitamin C group (P 0.5). At week 28, only 10
(18%) of 57 and 7 (14%) of 52 women had a cervicovaginal
infection (P 0.05). At week 36, only 10 women had infection
(6 in the placebo group and 4 in the vitamin C group; P 0.05).
There were no differences between groups in the incidence of
infection, and only one woman had a recurrent infection. The
microorganisms most frequently isolated were C. albicans
(10.5% of the cases) and G. vaginalis (5.3% of the cases).
According to the food-frequency questionnaire, the mean
daily vitamin C intake was 63 and 68 mg in the placebo and
vitamin C groups, respectively. In the placebo group, the 25th
and 75
th
percentile intakes were 38 and 135 mg/d, respectively;
corresponding values for the vitamin C group were 45 and 118
mg/d. There were no differences in dietary vitamin C intake
between groups. Citrus fruit, tomatoes, and chilies (hot peppers)
were the most important dietary sources of vitamin C. According
to the personal records kept by the subjects, 80% reported
adherence to the supplementation protocol (83% in placebo
group and 85% in the vitamin C group).
The overall mean plasma vitamin C concentration at week 20
of gestation was 0.76 0.28 mg/dL (43.15 15.9 mol/L).
Plasma vitamin Cconcentrations decreased similarly throughout
pregnancy in both groups (between-subjects differences: P
0.001), and they followed a significant (P 0.001) linear trend
with time (Table 2).
The mean leukocyte vitamin C concentration in the groups
diverged as gestation progressed (between-subjects differences:
P 0.001). The concentration increased significantly between
weeks 20 and 36 in the vitamin Csupplemented group, but it
decreased over the same period in the placebo group (between-
subjects differences: P 0.001; Table 2).
The incidence of PROM was 4 (8%) of 52 women in the
vitaminCsupplementedgroupand14(25%) of 57womeninthe
FIGURE 1. Flow chart for a randomized vitamin C trial.
TABLE 1
Characteristics and outcome variables
1
Placebo
group
(n 57)
Vitamin C
group
(n 52)
Characteristics of the mothers
Age (y) 27.4 7.7
2
27.5 7.4
Number of gestations (n) 2.3 1.2 2.4 1.2
Height (cm) 155.4 6.7 155.0 6.9
Pregestational BMI (kg/m
2
) 23.8 3.86 24.2 4.2
Schooling (y) 11.0 3.1 10.3 2.7
Married [n (%)] 35 (61) 33 (63)
Characteristics of the fetuses
Percentage of weight expected for
height and gestational age at week
20 (%)
109 24 104 12
Gestational age at prenatal care (wk) 20 3 20 5
Outcome results
Birth weight (g) 3015 629 3015 513
Gestational age at birth (wk) 38.0 3.1 38.5 2.0
Premature births [n (%)] 14 (24) 7 (13.4)
PROM [n (%)] 14 (24) 4 (7.6)
3
1
PROM, premature rupture of the chorioamniotic membrane.
2
x SD (all such values).
3
Significantly different from placebo group, P 0.018 (chi-square
test).
PREVENTION OF PREMATURE RUPTURE OF MEMBRANES 861

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placebo group (P 0.018). Vitamin C supplementation ap-
peared to have a protective effect of 74%and an RRof 0.26 (95%
CI: 0.078, 0.837).
The overall incidence of preterm labor was 21 (19%) in 109,
but there was no significant difference between groups (24% in
the placebo group and 13% in the vitamin C group; P 0.142).
Preterm labor occurred in 9 of 18 pregnancies complicated with
PROMand in 12 of 91 pregnancies not complicated with PROM
(RR: 6.58; 95% CI: 2.17, 19.89).
DISCUSSION
Although premature rupture of the membranes can have mul-
tiple causes, the mechanismthat triggers the final damage affects
the integrityof the amniochorionconnective tissue bydecreasing
the tissular content of collagen and causing the sudden loss of
mechanical strength (1, 4). We hypothesized that vitamin Csup-
plementation during pregnancy could prevent PROM by modu-
lating collagen metabolism and favoring its deposit in fetal tis-
sues, including the amniochorion membranes.
Many factors can alter the availability of vitamin C during
pregnancy, and, under some circumstances, it might be necessary
to adjust the current recommended daily dietary allowance. We
assessed vitamin C status by measuring its concentration in
plasma andleukocytes in2groups of pregnant women; one group
ingested 100 mg vitamin C/d and the other ingested placebo. The
plasma concentration of vitamin C did not differ significantly
between groups. A possible explanation for this fact is that the
range of dietary vitamin C intakes is very small (40230 mg
vitamin C/d), and the authors who documented a direct and
significant relation between vitamin C intake and its plasma
concentration explored vitamin C intake ranges from 0 to 2500
mg/d (8, 21, 22) In contrast, leukocyte concentrations increased
in the vitamin C group but decreased in the placebo group, and
leukocyte concentration is considered an indicator of the stored
amount of vitamin (13, 23). We measured plasma vitamin C
concentration in fasted subjects because its concentration in
plasma is coregulated with blood glucose by insulin (24).
Plasma vitamin C concentration decreased progressively dur-
ing gestation regardless of supplementation. This decrease most
likely reflects the effects of hemodilution (3, 25) and of active
transport of vitamin C to the fetus, which increases throughout
pregnancy (26). Leukocyte vitamin Cconcentration differed sig-
nificantly between groups. Because the 2 groups did not differ in
their dietaryintake of vitaminC, these changes most likelyreflect
the effect of supplementation, which suggests that the extra vi-
tamin C was taken up by the leukocyte compartment. Thus,
leukocyte vitamin Cconcentration might serve as a direct marker
of compliance.
The amount of vitamin C contained in the supplement tablet
(100 mg/d) is less than the highest intake limit of 2000 mg/d
established by the Institute of Medicine (27); this explains the
lack of adverse effects of supplementation in our study. A daily
dose of 100mgascorbate incombinationwiththe average dietary
intake of 65 mg vitamin C appears to be sufficient to maintain
leukocyte an ascorbic acid concentration of 18 g (102 nmol)/
10
8
cells, which has been shown to prevent PROM (3). In our
study, the incidence of PROM in the placebo group was 3 times
that in the supplemented group, which represents a 76% protec-
tive effect of vitamin Csupplementation. To our knowledge, this
is the first example of a beneficial effect of vitamin Cin lowering
the incidence of PROM.
Our data are consistent witha previous studyof 2000women
that found an inverse relation between vitamin C intake and the
incidence of PROM (28), although only diet and clinical cervi-
covaginal infection were evaluated. Another recent trial of vita-
min C supplementation during pregnancy failed to show any
beneficial effect on the incidence of preterm labor (29). How-
ever, the latter study used preterm labor as the primary outcome
and did not evaluate the effect of supplementation on the inci-
dence of PROM. Moreover, the dose of vitamin C was much
higher (500 mg/d) than that in our study, and the method of
evaluating vitamin C concentration was colorimetric assay.
Large doses of vitamin Ccan cause adverse reactions, such as
those associated with oxidative stress (30). Reactive oxygen
species can enhance collagen degradation in chorioamniotic
membranes (31), whichcaninterfere withthe protective effect on
the gene expression of metalloproteinases (eg, MMP-2) and can
lead to increased collagen degradation, a condition thought to be
an important mechanism in the genesis of PROM (3234). This
potential, undesirable effect must be taken into account when
establishing the supplement dose of vitamin C. We propose that
vitamin Cstatus could be used as a functional indicator of the risk
of PROMand that a leukocyte vitamin Cconcentration 18 g
(102 nmol)/10
8
cells is an indicator of adequate bioavailability of
the vitamin (35).
We found no relation between infection and PROM, although
38% of the women had cervicovaginal infections by week 20 of
gestation. We treated these infections and found no infection
relapses in the women.
In conclusion, our data support the concept that vitamin C
at dietary doses (100 mg/d) can prevent PROM. Because
PROM may trigger 40% of all preterm labor, supplementa-
tion could be a valuable tool in sustaining pregnancy to term.
TABLE 2
Concentration of vitamin C in plasma and leukocytes
1
Weeks of gestation
Placebo
group
(n 57)
Vitamin C
group
(n 52) P
2
Plasma (g/dL)
3
20 0.78 0.28 0.74 0.28
24 0.73 0.27 0.74 0.25
28 0.72 0.26 0.72 0.29
32 0.63 0.24 0.68 0.26
36 0.55 0.25 0.58 0.21
Leukocytes (g/10
8
cells)
4
20 17.50 6.15 17.26 6.05 0.835
24 17.28 5.79 18.98 6.30 0.145
28 17.23 6.50 21.29 8.22 0.005
32 15.79 6.47 21.56 7.47 0.001
36 15.23 6.12 22.17 8.73 0.001
1
All values are x SD.
2
Comparison between groups for each week of pregnancy.
3
1 g/dL 56.8 mol/L. Plasma vitamin C concentration: within-
subject difference (time), P0.001; between-subject difference, P0.760;
there was no significant time treatment interaction.
4
1 g/10
8
cells 5.7 nmol/10
8
cells. Leukocyte vitamin C concentra-
tion: within-subject difference, P 0.01; between-subject difference, P
0.001. P for interaction 0.001; P for time 0.001; for P for treatment
0.001. Two-factor ANOVA with Tukeys correction for repeated measure
(gestational weeks 2036).
862 CASANUEVA ET AL

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The next challenge is to design educational programs to pro-
mote adequate intakes of vitamin C through the consumption
of fruit and vegetables and not through the taking of supple-
ments during pregnancy.
We thank the volunteer women for their valuable participation.
EC assisted with the development of the research study protocol, coordi-
nated the study, performed the statistical analyses, interpreted the data, and
took the major responsibility for writing the manuscript. CRassisted with the
recruitment and study of the subjects and collected data. MC and RMM
performed the laboratory experiments and collected data. FP assisted with
coordination of the study and with writing the manuscript. PV assisted with
the obstetric evaluations of the subjects. FVOassisted in the development of
the research protocol, review of the literature, interpretation of the data, and
revision of the manuscript. None of the authors had a personal or professional
conflict of interest.
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