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ORIGINAL RESEARCH |
From the University of Toronto, Women’s College Hospital, and Mount Sinai Hospital, Toronto; Queen’s University, Kingston General Hospital, Kingston; and University of Ottawa, Ottawa Hospital, General Campus, Ottawa, Ontario, Canada.
Address reprint requests to: Mark C. Walker, MD, Department of Obstetrics and Gynecology, Ottawa Hospital, General Site, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada; E-mail: mwalker{at}ottawahospital.on.ca.
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ABSTRACT |
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 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: In this case-control study, women with preterm PROM (n = 46) were compared with healthy pregnant women matched for gestational age and vitamin supplementation. Measurements included fasting homocysteine, red blood cell folate, albumin, creatinine, and complete blood count. Dietary intake was determined by a food frequency questionnaire. Socioeconomic and demographic factors were recorded.
RESULTS: There were no differences between fasting homocysteine, red blood cell folate, and vitamin B12 levels anddietary intake between patients and controls. There was lower hemoglobin in women with preterm PROM compared with the controls (P < .001). There was a three-fold increased odds of having a total family income of less than $25,000 (Canadian) (odds ratio 3.1, 95% confidence interval 1.6, 6) in women with preterm PROM.
CONCLUSION: Preterm PROM is associated with low maternal hemoglobin and low socioeconomic status. There was no association with nutritional intake. The lower hemoglobin level may be a marker for subclinical infection.
Up to 4% of all pregnancies are complicated by preterm premature rupture of membranes (PROM).1 A third of all preterm births are a result of preterm PROM, and it is the number one identifiable cause of preterm delivery.2 There is significant perinatal morbidity and mortality associated with preterm PROM caused by prematurity, infection, and pulmonary hypoplasia.3
There are a number of risk factors, which have been associated with preterm PROM, including antepartum bleeding, cigarette smoking, previous preterm PROM or preterm delivery, low socioeconomic status, lower genital tract infection, multiple gestation, and polyhydramnios.4–6 The etiology of preterm PROM is believed to be multifactorial.2
Factors that alter collagen structure and interlinking architecture have been associated with preterm PROM. It has been suggested that pregnant women with infants born with connective tissue disorders such as Ehlers-Danlos syndrome are at increased risk of preterm PROM.7 Nutritional deficiencies that affect collagen formation have been shown to alter collagen structure.2 The strength of collagen is maintained through its crosslinks, which are formed through a series of reactions mediated by lysyl oxidase.8 Lysyl-oxidase is a copperdependent enzyme. Women with preterm PROM have been found to have lower copper levels than women in preterm labor.9 Vitamin C is a cofactor for proline hydroxylation and is essential for the formation of the triple helical structure of collagen.2 Studies have shown an association between low vitamin C levels and preterm PROM.10
High levels of homocysteine have also been associated with abnormalities in collagen cross-linking.11–13 Hyperhomocysteinemia can occur because of genetic or acquired nutritional deficiencies. The most common causes of mild hyperhomocysteinemia are the result of thermolabile methylenetetrahydrofolate reductase mutation and vitamin B12 and folate deficiencies.14,15 Vitamin B12 and folate are important cofactors in homocysteinemetabolism. Homozygosity for the thermolabile methylenetetrahydrofolate reductase mutation results in decrease of methylenetetrahydrofolate reductase activity, which leads to mildly elevated homocysteine when dietary folate is low.16
To date, there are no studies that have looked at the association of preterm PROM to poor nutritional status using biochemical markers and dietary intake of micro-nutrients. Studies have compared prepregnancy weight and weight gain during pregnancy to assess nutritional status.17 Studies that have found an association between low socioeconomic status and preterm PROM have assumed that low socioeconomic status reflects poor nutritional status in these women.5,18
The primary objective of this case-control study was to estimate if pregnant women with preterm PROM have higher serum levels of homocysteine compared with normal pregnant women. We hypothesized that these higher levels of homocysteine would reflect nutritional deficiencies of serum folate and vitamin B12. Secondary objectives were to evaluate whether pregnant women with preterm PROM had other nutritional deficiencies with respect to caloric intake, fat and protein consumption, and vitamin intake. Socioeconomic status as determined by education and family income was assessed as a cofactor in preterm PROM.
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MATERIALS AND METHODS |
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Women were eligible to be subjects for this study if they had objective evidence of preterm PROM by both positive ferning and nitrazine test at the time of admission by sterile speculum examination. They were only eligible if the history of preterm PROM had occurred within 12 hours of presenting to these institutions.
Women with preterm PROM were excluded if they had evidence of active labor 2 hours before or after they had ruptured their membranes. Active labor was defined as painful uterine contractions with progressive dilatation and effacement of the cervix. They were also excluded if they had evidence of chorioamnionitis, which was determined by evidence of maternal fever and fetal tachycardia. Women with preterm PROM were also excluded if they delivered before the fasting bloodwork was obtained or if they had renal disease or known folateor vitamin B12 deficiency. Women with risk factors forpreterm PROM, such as multiple gestation, polyhydramnios, cervical cerclage, or leakage of fluid after amniocentesis, were also excluded.
Signed informed consent was obtained from each patient. Upon entering the study, they had a sterile speculum examination to confirm rupture of membranes and had cervical cultures for Ureaplasma urealyticum and vaginal cultures for bacterial vaginosis. They were asked to fast after midnight until 8:00 AM. At this time, maternal blood was taken for homocysteine, complete bloodcount, red blood cell folate, vitamin B12, albumin, andcreatinine. At the time of delivery, umbilical artery and vein blood were taken to assess fetal homocysteine levels.
The control population, which was matched to the patients by gestational age within 3 weeks and for vitamin supplementation, did not have vaginal or cervical cultures obtained. We concluded that this additional vaginal examination would deter women from participating in this study. Maternal fasting bloodwork was obtained at the appropriate gestational age to the patient to whom they were matched. Fetal umbilical artery and vein blood for homocysteine levels were obtained at the time of delivery for the control population.
Women in the case and control populations were asked to complete a self-administered food frequency questionnaire, called the Block Healthy Habits and History questionnaire (DIETSYS 3.0, National Cancer Institute, Bethesda, MD).19 This program collates information from dietary consumption surveys and calculates total daily dietary intake of vitamins, minerals, fats, etc. Data were also obtained on past medical and obstetric histories.
Fasting venous blood was drawn from the antecubital vein or from the hand. Homocysteine and red blood cellfolate were collected in K2 ethylenediaminetetraaceticacid Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ). Homocysteine specimens were put on a cold pack (4C) immediately after collection, transported to the laboratory within 30 minutes, and centrifuged at 4C for 10 minutes at 3000 xg. Plasma was removed and stored at -20C until analysis. Samples were assayed in batches.
Blood for vitamin B12, albumin, and creatinine werecollected in serum separator tubes. Complete blood count, red blood cell folate, serum B12, serum creatinine, and albumin were measured with standard laboratory procedures. Total homocysteine was determined by high-performance liquid chromatography with electrochemical detection and pulsed integrated amperometry (Dionex DX-500 Ion Chromatograph; Dionex, Oakville, Ontario, Canada).20
A sample size calculation had determined that 40 patients and 40 controls would be required for a minimally detectable difference of 2 µmol/L of homocysteine with an 
of 0.05 and a ß of 0.2 and a standard deviation of 2.5 based on previously published work, which demonstrated that homocysteine levels decrease with advancing gestation.21
The primary outcome of plasma homocysteine was compared between groups with the Student t test. All other continuous variables were compared in this manner. All dichotomous outcomes were compared with the 
2 test. The Fisher exact test was used where appropriate. We did not use the paired Student t test or McNemar test as we wanted a more conservative approach because of complexities in the matching process. Logistic regression was used to identify independent predicators of preterm PROM. Statistical significance was set at P < .05 for the primary outcome. For secondary outcomes, significance was set at P < .001 as an adjustment for multiple statistical testing.
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RESULTS |
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Ninety-one percent of women with preterm PROM took prenatal vitamins at some time during their pregnancy, and up to 74% of these women took prenatal vitamins consistently starting before 12 weeks’ gestational age.
There were no significant differences between patients and controls for all baseline demographic features, including age, race, gravidity, previous or present pregnancy complications, or maternal medical history (Tables 1
and 2). There were fewer previous term deliveries in women with preterm PROM compared with the control population (Table 1). There were no significant differences between the patient and control groups for socioeconomic factors such as smoking, alcohol consumption, marital status, obstetric care provider, or planned pregnancy (Table 3). There was a significant difference between women with preterm PROM and their matched controls for total family income and education when using univariate analysis (Table 3). The crude odds ratio for women with preterm PROM, having a total family income less than $25,000 was 6.94 (95% confidence interval [CI] 1.67, 33.2) and for having a lower education was 6.16 (95% CI 1.7, 24.3). Using logistic regression, it was determined that total family income less than $25,000 was the most important factor related to preterm PROM with an adjusted odds ratio of 3.1 (95% CI 1.6, 6.0).
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). There were also no significant differences in dietary intake of nutrients including calcium, thiamin, riboflavin, niacin, vitamin C, folate, vitamin E, zinc, vitamin B6, and - or ß-carotene (Table 4).
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). The mean hemoglobin for women with preterm PROM was 113.5 g/L compared with 122.4 g/L in women without preterm PROM (Table 5). There was a four-fold increased odds of having preterm PROM in women with hemoglobin less than 111g/L (odds ratio 4.33, 95% CI 1.38, 14.17). There was no difference in mean corpuscular volume between patients and controls (Table 5). There was also a significant decrease in albumin and increase in white blood cell count in the case population compared with the controls (Table 5). There were no significant differences between women with and without preterm PROMfor platelets, creatinine, vitamin B12, red blood cell folate, and fasting homocysteine (Table 5).
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). However, there were higher umbilical artery homocysteine levels in neonates born at term from women without preterm PROM compared with those born preterm to women with preterm PROM (Figure 1). This higher level of homocysteine was also observed in umbilical vein levels of neonates born to women without preterm PROM (Figure 1).
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DISCUSSION |
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Our study did not confirm a decreasing gradient in homocysteine levels from neonatal umbilical vein to neonatal umbilical artery in preterm or term infants. This lack of a gradient between the umbilical vein does not support the previous findings by Malinow et al.23 There were, however, higher levels of homocysteine in the umbilical artery and vein of term infants compared with preterm infants. Up to 37 weeks, the weight of the fetus increases in a linear fashion at which time the growth rate slows.24 Methionine acts as a methyl donor in deoxyribonucleic acid and ribonucleic acid synthesis.25 We hypothesize that at earlier gestation there is a greater number of dividing cells, and, therefore, there is a greater requirement for methionine for methylation of deoxyribonucleic acid and ribonucleic acid. This would lead to lower homocysteine levels as we observed. Furthermore, at term, the growth rate of the fetus is reduced, and, therefore, the fetus requires less methionine, which results in higher homocysteine levels.
In this study, maternal hemoglobin is lower in pregnant women with preterm PROM compared with normal pregnant women. There have been multiple studies showing an association between maternal anemia and preterm birth and low birth weight.18,26–29 These studieshave significant limitations. Most of these studies are retrospective obtaining data from large registries.28,29 None of the studies differentiates preterm labor from preterm PROM as a cause of preterm delivery.18,26–29 Many of these studies do not determine cause of maternal anemia, and many hypothesize that it is related to underlying nutritional or social deprivation.5,18,29 One prospective cohort study stratified women for the type of anemia.26 They found a five-fold increase in preterm birth for women with iron deficiency anemia and a two-fold increase in preterm birth for women with other types of anemia compared with nonanemic controls.26 There is one case-control study that looked at risk factors associated with preterm PROM and found no difference in maternal hemoglobin between patients and controls.17
Our study is the first to show an association between preterm PROM and low maternal hemoglobin. We found a four-fold increased risk of having preterm PROM in women with hemoglobin less than 111 g/L. There was no difference in the mean corpuscular volume between women with preterm PROM and their matched controls. The mean corpuscular volume was found to be in the normocytic range, which is more consistent with anemia of chronic disease than with iron deficiency anemia.30 These women are unlikely to be iron deficient because 72% were on vitamin supplementation starting before 12 weeks’ gestational age. As well, these women have no evidence of other nutrient deficiencies. A limitation of this study is that we do not have serum ferritin levels to differentiate the type of anemia. This lower hemoglobin and lower serum albumin level in women with preterm PROM may be a marker of subclinical infection or inflammatory process.30
Women with preterm PROM were excluded from this study if they had overt signs of chorioamnionitis such as maternal fever or maternal or fetal tachycardia. There was a significant increase in maternal white blood cell count in women with preterm PROM compared with controls. However, this was found to be secondary to glucocorticoid administration to help with fetal lung maturity, which was universally given as the standard of care.31,32 Only 9% of women with preterm PROM had a positive culture for group B streptococcus, and there were no positive cultures for bacterial vaginosis or Trichomonas. Of the 21 women who had cervical cultures for Ureaplasma urealyticum, 66.7% were positive. We were unable to obtain samples from the control population and, therefore, could not make a direct comparison. However, this is the rate of colonization that has been found in the general pregnant population without pregnancy complications.33 Lower maternal hemoglobin in women with preterm PROM may be the result of subclinical infection secondary to normal endogenous vaginal flora, which we were not able to detect. It is also conceivable that hydration of these patients may have lowered hemoglobin levels. However, the small amount over the short period before blood was drawn is unlikely to have impacted in an important way.
We found a three-fold increased risk of having preterm PROM in pregnant women with total family income less than $25,000. This relationship between low socioeconomic status and preterm PROM has been reported in the past and was found to be related to inequalities in access to health care, poor nutritional status, and rate of genital tract colonization.5,18 In our study, other markers of low socioeconomic status such as smoking, access to antenatal care, unplanned pregnancy, marital status, and alcohol consumption were similar between women with preterm PROM and controls. Our study specifically looked at nutritional status using both biochemical markers and a validated dietary questionnaire.
In summary, we did not find high levels of homocysteine associated with preterm PROM. We did find significantly lower maternal hemoglobin in women with preterm PROM compared with controls and conclude this may represent the inflammatory response associated with preterm PROM or subclinical infection. We did find women with preterm PROM were more likely to have lower socioeconomic status represented by low family income. However, these women did not have evidence of nutritional deficiencies when measured in the serum or when determined by dietary intake. Perhaps other factors associated with lower socioeconomic status, such as stress, depression, and poor general health are cofactors and place women of lower economic status at risk for preterm PROM. These would be important variables for study in future research on preterm PROM.
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Footnotes |
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Received December 18, 2001. Received in revised form May 3, 2002. Accepted June 6, 2002.
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REFERENCES |
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