- Click here for explanation of classification scheme.

To investigate the association of maternal plasma acid concentration with subsequent risk of gestational diabetes.

• initiated pregnancy before 16 weeks of gestation
• nulliparious

• women who experienced a spontaneous abortion or other fetal demise or who induced abortion
• those women for whom the outcome of pregnancy was unknown because the woman moved, delivered elsewhere, or were missing medical records.
• women with pregestational diabetes mellitus

Recruitment : The population was drawn from participants of the ongoing OMEGA Study(Washington State), a prospective cohort study of maternal dietary risk factors of preeclampsia. The researchers primarily approached and enrolled nulliparous women.

Design : prospective cohort

Blinding used -All assays were performed without knowledge of pregnancy outcome.

Intervention:  not applicable

Statistical Analysis :

• continuous variables were presented as means ± standard error of mean (SE)
• women were grouped according to quartiles determined by the distribution of plasma ascorbic acid concnetrations among the entire cohort
• created a dichotomous variable for dietary vitamin C intake using the Institute of Medicine estimated average requirement for pregnant women (<70 vs. >= 70 mg/day-EAR) as the cutoff value
• frequency distributions were utilized to examine maternal sociodemographic characteristics and medical and reproductive histories according to plasma ascorbic acid concentrations
• generalized linear models were fitted, using a log-link function. These were utilized to derive risk ratios (RRs) and 95% confidence intervals (CIs)
• explored the possibility of nonlinear relation between plasma ascorbic acid concentrations and diabetes risk using generalized additive modeling (GAM) procedures by using S-PLUS (version 6.1; Insightful Corp., Seattle, WA)
• multivariate analysis-evaluated linear trends in risk by treating 4 quartiles of plasma ascorbic acid concentrations as a continuous variable after assigning a score (1, 2, 3, and 4) as its value.
• dietary vitamin C was adjusted for total energy intake
• to assess confounding, variables were entered into a generalized linear model. Final generalized linear models included covariates that altered unadjusted risk ratios by at least 10%, as well as those covariates of a priori interest (eg, advanced maternal age and prepregnancy adiposity).

 

Timing of Measurements

• Maternal age was determined at the time of the interview.
• Nonfasting blood samples were collected in EDTA 10-ml Vacutainer tubes at 13 weeks gestation, on average (interquartile range =11-16 weeks gestation). 
• Maternal and infant medical records were reviewed  approximately 7-9 months after participants were enrolled in the study to collect detailed information concerning antepartum, labor and delivery characteristics, and conditions of the newborn.

Dependent Variables

Maternal plasma ascorbic acid

• Nonfasting blood samples were collected in EDTA 10-ml Vacutainer tubes at 13 weeks gestation, on average (interquartile range =11-16 weeks gestation. 
• The samples were protected from ultraviolet light , kept on wet ice, and processed within 20 minutes of phlebotomy. The median time between participants' last meal and phlebotomy was 2.5 for both cases and controls.
• Plasma decanted into cryovials was preserved with metaphosphoric acid/dithiothreitol solution frozen at -80 degrees until analysis.
• Laboratory analyses were performed within 24 months of blood collection. Ascorbic acid concentrations have been shown to be stable for more than 57 months under these storage conditions.
• Plasma ascorbic acid (ie, total ascorbate) was determined on a Roche Cobas(Branchburg, NJ) Mira Plus Chemistry analyzer using a colorimetric procedure.
• The intra-and interassay coefficients of variation for the assay used were both less than 10%.

Pregnancy Outcome

• Maternal and infant medical records were reviewed approximately 7-9 months after participants were enrolled in the study to collect detailed information concerning antepartum, labor and delivery characteristics, and conditions of the newborn.
• Laboratory results were extracted from medical records for participants' 50-g 1-hour oral glucose tolerance test and from the diagnostic 100-g 3-hour oral glucose tolerance test.
• Women were classified as having a pregnancy complicated by gestational diabetes if results from their diagnostic test met the then-current National Diabetes Data Group criteria. Women were classified as having gestational diabetes mellitus if 2 or more of the following plasma glucose concentrations were abnormal according to these criteria: fasting>= 105 mg/dl, 1-hour >=190 mg/dL, 2-hour>=165 mg/dl, 3-hour>=145 mg/dL.

Independent Variables

• maternal age*
• height
• reproductive and medical history
• race*
• age*
• smoking during pregnancy*
• first degree family history of type 2 diabetes*
• prepregnancy adiposity*- prepregnancy body mass index (BMI), used as a measure of overall maternal adiposity, was calculated as weight (kg) divided by height (m) squared. All participants reported their maximum height and weight 3 months before the index pregnancy
• annnual household income*
• parity was reported as the number of previous pregnancies lasting beyond 20 weeks of pregnancy.
• Maternal habitual dietary intake during the peri period and early pregnancy were ascertained at 12 weeks gestation, on average , using the self-administered , 121 item semiquantitative food frequency questionnaire developed for the Women's Health Initiative Clinical Trial. Food composition values for vitamin C and other nutrients were obtained from the University of Minnesota Nutrition Coding Center nutrient data base (Nutrition Coordinating Center), Minneapolis, MN. 

* possible confounders

Control Variables

 

Initial N: 755

Attrition (final N):  755

Age: see Table 1

Ethnicity: see Results

Other relevant demographics: see Results 

Anthropometrics

Location: Department of Epidemiology, University of Washington School of Public Health and Community Medicine, Seattle, Washington.

 

Table 1 shows the characteristics of members of the study cohort according to quartile of plasma ascorbic acid concentrations. Overall, participants included in the analysis were primarily white, well-educated, and employed during pregnancy.

Table 1 Characteristics* of Study Cohort Members (n=755) According to Maternal Plasma Ascorbic Concentrations Measured in Early Pregnancy, Seattle and Tacoma, Washington , USA 1996-2000. 

 

-	Quartiles of >	Maternal Plasma >	Ascorbic Acid>	Concentrations
-	-	(µmol/L)	-	-
-	1(Low)	2	3	(4 High)
-	(n=189)	(n=189)	(n=189)	(n=188)
Plasma ascorbic acid concentrations	-	-	-	-
Quartile mean±SE	46.7±0.6	60.8±0.2	69.2±0.2	85.0±0.7
Quartile median	49.8	61.2	68.9	82.2
Quartile range	11.2-55.9	56.0-65.0	65.1-74.5	74.6±128.5
Dietary vitamin c intake(mg/d);mean±SE	-	-	-	-
Maternal age(y)	113.8±4.7	125.2±5.1	127.9±5.0	138.2±0.7
<20	2(1)	1(1)	2(1)	3(2)
20-34	133(70)	134(71)	142(75)	135(72)
35-39	45(23)	42(22)	40(21)	41(22)
>=40	9(5)	12(6)	5(3)	9(5)
Maternal Race/Ethnicity	-	-	-	-
European ancestry	140(74)	161(86)	171(90)	167(89)
African ancestry	6(3)	5(3)	2(1)	6(3)
Asian ancestry	26(14)	13(7)	8(4)	6(3)
Other	17(9)	9(5)	8(4)	9(5)
Unknown	-	1	-	-
Unmarried	23(12)	19(10)	25(13)	27(14)
=< 12 years education	9(5)	5(3)	9(5)	9(5)
Nulliparous	150(79)	167(88)	168(89)	169(90)
No prenatal vitamins	10(5)	2(1)	5(3)	5(3)
Smoked in pregnancy	12(6)	13(7)	9(5)	11(6)
First degree family history of diabetes	35(18)	27(14)	168(89)	25(13)
Pre-pregnancy body mass index( kg/m2)	-	-	-	-
<20.0	38(20)	35(19)	5(3)	43(23)
20.0-24.9	89(47)	108(57)	9(5)	105(56)
25.0-29.9	31(16)	29(15)	20(11)	32(17)
>= 30.00	31(16)	17(9)	44(23)	8(4)
Worked during pregnancy	168(89)	171(91)	103(55)	172(92)
Annual household income  ( US $)	-	-	-	-
<30,000	8(4)	10(5)	10(6)	8(4)
30,000-69,999	51(27)	58(38)	58(32)	66(36)
>= 70,000	127(68)	118(63)	115(63)	108(59)
Not reported	3	3	6	6
Calendar month at blood collections	-	-	-	-
March-May	44(23)	44(24)	49(26)	63(34)
June-August	41(22)	46(24)	50(27)	39(21)
September-November	48(25)	51(27)	39(21)	39(21)
December-February	56(30)	48(25)	51(27)	47(25)
Gestational age at blood collection(weeks) ; mean ±SE	13.3±0.3	13.0±0.3	13.4±0.3	13.2±0.2
*Number(%) except where indicated

Women with plasma ascorbic concentrations less than 55.9 µmol/L (lowest quartile)experienced a 3.4 -fold increased risk of diabetes (CI=1.2-9.2) as compared with women whose plasma concentrations were 74.6 µmol/L or higher (highest quartile) (Table 2).

Table 2  Association of Gestational Diabetes Mellitus (based on 1997 NDDG Criteria) with Maternal Plasma Ascorbic Acid Concentrations Measured in Early Pregnancy

 

 

-	Quartiles of >	Maternal Plasma >	Ascorbic Acid>	Concentrations
-	-	(µmol/L)	-	-
-	1(Low)	2	3	(4 High)*
-	(n=189)	(n=189)	(n=189)	(n=188)
Quartile median	49.8	61.2	68.9	82.2
Quatile range	11.2-55.9	56.0-65.0	65.1-74.5	74.6-128.5
Cases; no. (%)	17(9.0)	7(3.7)	4(2.1)	5(2.7)
RR(95% CI)	3.4(1.2-9.2)	1.4(0.4-4.4)	0.8(0.2-2.9)	1.0
Adj. RR¶  (95% CI)	3.1(1.0-9.7)	1.6(0.5-5.5)	0.8(0.2-3.5)	1.0
Adj. RR € (95% CI)	2.9(0.9-9.2)	1.6(0.5-5.4)	0.8(0.2-3.4)	1.0

NDDG indicates National Diabets Data Group * Reference category

¶ Adjusted for maternal age (continous) , race/ethnicity, first degree family history of diabetes, parity, pre-pregnancy body mass index (grouped linear), and annual household income.

€ Adjusted for same variable as in first adjustment model, plus dietary fiber intake(continous).

Women whose daily vitamin C intake was <70 mg, as compared with those consuming more vitamin C experienced a 2.2-fold increased risk of diabetes (95% CI=1.0-4.6) (Table 3). 

Table 3 Asssociation of Gestational Diabetes Mellitus with Maternal Daily Dietary Vitamin C Intake

 

-	Dietary Vitamin C>>	>>>Intake*♦ (mg/d)
-	<70(n=153)	>= 70¶ (N=576)
Cases; no.(%)	11(7.2)	19(3.3)
RR(95% CI)	2.2(1.0-4.6)	1.0
Adj. RR ¶(95% CI)	1.8(0.8-4.4)	1.0

* based on 729 women (30 cases).

♦ A dichotomous variable for dietary vitamin C intake was created using the Institute of Medicine estimated average requirement for pregnant women (<70 vs. >70 mg/d) as the cut-off value.

§ Reference Category

¶ Adjusted for maternal age (continous) , race/ethnicity, family history of type 2 diabetes, parity, pre-pregnancy body mass index (grouped linear), annual household income, and total calories intake (continous).

Inferences regarding the relatinship between maternal ascorbic acid concentrations and gestational diabetes risk were largely similar in direction and magnitude when analyses were repeated using the less stringent diagnostic criteria that was being advocated by the NDDG at the time of the study(Table 4).

Table 4  Association of Gestational Diabetes Mellitus (Based on 2002 NDDG Criteria ) with   Maternal Plasma Ascorbic Acid Concentrations Measured in Early Pregnancy

 

-	Quartiles of >	Maternal Plasma >	Ascorbic Acid>	Concentrations
-	1(Low)	2	3	(4 High)*
Quartile Median	49.8	61.2	68.9	82.2
Quartile Range	11.2-55.9	56.0-65.0	65.1-74.5	74.6-128.5
Cases;No. (%)	19(10.0)	11(5.8)	5 (2.6)	6(3.2)
Total Population, N	189	189	189	188
RR (95% CI)	3.2(1.3-7.7)	1.8(0.7-4.8)	0.9(0.3-2.8)	1.0
Adj RR¶(95% CI)	2.9(1.1-7.9)	1.7(0.6-4.9)	0.8(0.3-3.1)	1.0
Adj RR € (95% CI)	2.8(1.0-7.7)	1.7(0.6-4.8)	0.8(0.3-3.0)	1.0

NDDG indicates National Diabets Data Group * Reference category

¶ Adjusted for maternal age (continous) , race/ethnicity, first degree family history of diabetes, parity, pre-pregnancy body mass index (grouped linear), and annual household income.

€ Adjusted for same variable as in first adjustment model, plus dietary fiber intake(continous).

Other Findings

The researchers noted that the generalized additive model resulted in an approximately inverse  linear relationsship between the log-odds of diabetes risk and plasma ascorbic acid, particularly among those women with concentrations between 40 and 100 µmol/L.

In this prospective cohort of pregnant women, the researchers observed an inverse relationship between plasma ascorbic acid concentrations and incidence of gestational diabetes mellitus.

Concerns remain about the paucity of empiric evidence concerning dietary patterns associated with optimal pregnancy outcomes.

Limitations:

1. Missclassification of maternal vitamin C intake (or plasma ascorbic acid concentrations) could have resulted in diluting their associations with diabetes.

2. Inability to assess the independent effects of multivitamin use and diabetes risk because of the near-universal use of prenatal vitamins by the study population.

3. Researchers could not exclude the possibility of residual confounding.

4. The relatively small size of the study cohort, the estimated effect measures were imprecise.

5. The small sample size also impeded the efforts to examine the risk of diabetes in relation to maternal dietary intake patterns.

6. Another concern relates to the misclassification of maternal anthropometric status.

7. Generaliizability of results could be limited. The study focused primarily on nulliparous women, and cohort members were predominantly white, well-educated women.

 

The limitations and critique of the study, as stated by the authors appear to be very appropriate.

Analytical longitudinal surveys refer to what epidemiologists term prospective or cohort studies. A Cohort Study is a study in which patients who presently have a certain condition and/or receive a particular treatment are followed over time and compared with another group who are not affected by the condition under investigation. Studies of this kind provide a better opportunity than one time cross sectional studies to examine whether certain behaviors do in fact lead to (or cause) the disease.