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Objective This study was undertaken to validate a self-administered questionnaire in verifying the diagnosis of preeclampsia, eclampsia, or toxemia in a group of women with a greater than 20-year history of preeclampsia. Study Design Questionnaires were mailed to a random sample of 144 women who received a diagnosis of any of these 3 conditions and 158 women who had normotensive pregnancies at Mayo Clinic, Rochester, Minnesota, from 1960-1979. Results A previous diagnosis of preeclampsia, eclampsia, or toxemia was verified with 80% sensitivity and 96% specificity. Conclusion Our validated questionnaire may be a useful research tool in identifying women with a previous history of preeclampsia. Women with a history of preeclampsia had a higher prevalence of future hypertension than those with a history of normotensive pregnancy.

Background A history of preeclampsia is an independent risk factor for cardiac events and stroke. Changes in vasculature structure that contribute to these associations are not well understood. Objective The aim of this study was to quantify coronary artery calcification (CAC), a known risk factor for cardiac events, in a prospective cohort of women with and without histories of preeclampsia. Study Design Women without prior cardiovascular events (40 with and 40 without histories of preeclampsia, matched for parity and age at index birth) were recruited from a large population-based cohort of women who were residents of Olmsted County, Minnesota, and who delivered from 1976 through 1982. Computed tomography was performed to measure CAC in Agatston units. All pregnancy histories and covariates were confirmed by review of the medical records. Current clinical variables were assessed at the time of imaging. Differences between women with and without histories of preeclampsia were examined using χ 2 tests and tests; CAC, in particular, was compared as a categorical and ordinal variable, with a χ 2 test and with Wilcoxon 2-sample tests and ordinal logistic regression, as appropriate. Results Mean age (SD) at imaging was 59.5 (±4.6) years. Systolic and diastolic blood pressures, hyperlipidemia, and current diabetes status did not differ between women with and without histories of preeclampsia. However, the frequencies of having a current clinical diagnosis of hypertension (60% vs 20%, P 2 (expressed as median [25th-75th percentile], 29.8 [25.9-33.7] vs 25.3 [23.1-32.0], P = .023) were both greater in the women with histories of preeclampsia compared to those without. The frequency of a CAC score >50 Agatston units was also greater in the preeclampsia group (23% vs 0%, P = .001). Compared to women without preeclampsia, the odds of having a higher CAC score was 3.54 (confidence interval [CI], 1.39–9.02) times greater in women with prior preeclampsia without adjustment, and 2.61 (CI, 0.95–7.14) times greater after adjustment for current hypertension. After adjustment for body mass index alone, the odds of having a higher CAC based on a history of preeclampsia remained significant at 3.20 (CI, 1.21–8.49). Conclusion In this first prospective cohort study with confirmation of preeclampsia by medical record review, a history of preeclampsia is associated with an increased risk of CAC >30 years after affected pregnancies, even after controlling individually for traditional risk factors. A history of preeclampsia should be considered in risk assessment when initiating primary prevention strategies to reduce cardiovascular disease in women. Among women with histories of preeclampsia, the presence of CAC may be able to identify those at a particularly high cardiovascular risk, and should be the subject of future studies.

OBJECTIVE: Pregnancy is associated with significant change in leukocyte composition/function in the establishment/adaptation to pregnancy and parturition. We characterized methylation profiles longitudinally in a group of healthy pregnant women. STUDY DESIGN: The methylation profiles were characterized in maternal buffy coat DNA longitudinally in pregnancy: early (

Objective Hyperhomocysteinemia is associated with an elevated cardiovascular disease risk. We examined whether women with a history of hypertension in pregnancy are more likely to have a high level of serum homocysteine decades after pregnancy. Study Design Serum homocysteine was measured at a mean age of 60 years in nulliparous women (n = 216), and women with a history of normotensive (n = 1825) or hypertensive (n = 401) pregnancies who participated in the Genetic Epidemiology Network of Arteriopathy (GENOA) study. Relationships between homocysteine and pregnancy history were examined by linear and logistic regression, controlling for multiple covariates including personal and family history of hypertension, diabetes, obesity, tobacco use, and demographics. Results A history of hypertension in pregnancy, when compared with normotensive pregnancy, was associated with a 4.5% higher serum homocysteine level ( P = .015) and 1.60-fold increased odds of having an elevated homocysteine (95% confidence interval, 1.15–2.21; P = .005) after adjusting for potentially confounding covariates. In contrast, a history of normotensive pregnancy, as compared with nulliparity, was associated with a 6.1% lower serum homocysteine level ( P = .005) and a 0.49-fold reduced odds of elevated homocysteine levels (95% confidence interval, 0.32–0.74; P Conclusion Homocysteine levels decades after pregnancy are higher in women with a history of pregnancy hypertension, even after controlling for potential confounders. Thus, pregnancy history may prompt homocysteine assessment and risk modification in an attempt at primary prevention of cardiovascular disease.

novel association with lower maternal serum levels in preeclampsia at delivery Wendy White, Jonathan O’Brien, Elizabeth Baldwin, Arij Faksh, Angelica Garrett, Brian Brost, Carl Rose, Norman Davies, Zhifu Sun, Stephen Turner, Vesna Garovic Mayo Clinic College of Medicine, Maternal-Fetal Medicine, Rochester, MN, Mayo Clinic College of Medicine, Biomedical Statistics and Informatics, Rochester, MN, Mayo Clinic College of Medicine, Hypertension and Nephrology, Rochester, MN OBJECTIVE: Eotaxin-3 or CCL26 is a chemokine important in leukocyte recruitment implicated in vasculitis, atopic dermatitis, and asthma, but has not been described in the context of normal or pathologic pregnancy. DNA methylation can inversely correlate with gene transcription. We sought to characterize methylation patterns in the CCL26 gene in normal and preeclamptic pregnancy and correlate with serum protein levels. STUDY DESIGN: The methylation profile of 2 CpG sites in the CCL26 gene was characterized in DNA from maternal blood longitudinally in 14 women at 16 gestational weeks, at delivery and postpartum; in 14 nulligravid controls; and in 14 preeclamptics at delivery. All women were non-smokers, matched for age and BMI. Genomic DNA was derived from buffy coat, purified, and bisulfite modified then run on the Illumina platform. Mean methylation levels at each CpG site and protein levels in serum as determined by ELISA were compared with a t-test. RESULTS: CCL26 methylation patterns did not differ significantly between the non-pregnant groups. In contrast, CCL26 CpG sites were significantly hypo-methylated during early pregnancy as compared with the non-pregnant state (p 10-3) but hypermethylation in preeclamptic women at delivery (p 10-3). ELISA demonstrated decreased levels of CCL26 in maternal serum in preeclampsia compared with normal pregnancy at the time of delivery (0.86 v. 1.69 pg/ml; p 0.03). CONCLUSION: Our results demonstrate that early pregnancy is associated with decreased methylation and preeclampsia at the time of delivery correlates with relative hypermethylation in the CCL26 gene. Methylation levels inversely correlate with a significant decrease in protein level as measured by ELISA in serum of women with preeclampsia. Altered methylation may be an epigenetic mechanism to control CCL26 production in normal early pregnancy and in preeclampsia. Methylation analysis can identify novel chemokines involved in normal and pathologic pregnancy.

in IL1 gene family in normal pregnancy Wendy White, Brian Brost, Jonathan O’Brien, Elizabeth Baldwin, William Watson, Carl Rose, Norman Davies, Zhifu Sun, Stephen Turner, Ramakrishna Edukula, Vesna Garovic Mayo Clinic College of Medicine, Maternal Fetal Medicine, Rochester, MN, Mayo Clinic College of Medicine, Biomedical Statistics and Informatics, Rochester, MN, Mayo Clinic College of Medicine, Nephrology and Hypertension, Rochester, MN OBJECTIVE: The Interleukin 1 family contains Interleukin 1alpha (IL1a), Interleukin 1beta (IL1b), IL1 receptor antagonist (IL1RA) and two receptors ILR1 and 2. These are important proinflammatory mediators in the establishment/ maintenance of normal pregnancy. STUDY DESIGN: The methylation profiles of the IL1 family (10 CpG sites in 5 genes) were characterized in maternal DNA from blood longitudinally in 14 women at 16 weeks, at delivery and postpartum (n 42) and in 14 nulligravid women. All patients were non-smokers, matched for age and BMI. Genomic DNA was derived from buffy coat, purified, bisulfite modified and run on the Illumina Methylation Assay platform. Mean methylation levels at each site were compared using a Students or paired t-test. Serum IL1b levels were measured by high sensitivity ELISA. RESULTS: The mean beta values (methylation %) at 10 CpG sites in 5 IL1 genes are shown for each time point and for the nulligravid controls in the table below; values in bold represent statistically significant differences between pregnant and non-pregnant groups. Methylation patterns are similar in both non-pregnant groups postpartum and nulligravid. Methylation levels in the ILRA and IL1R1 do not change with pregnancy. IL1a, IL1b, and IL1R2 become less methylated in early pregnancy compared with postpartum levels (p 0.0005; p 0.0007; p 0.004/p 0.007) or nulligravid controls (p 0.03; p 0.006; p 0.00002/p 0.02) and hypomethylation persists or increases at delivery. Serum IL1 beta levels are higher in early pregnancy (mean 0.108 pg/mL) compared with normal values from the literature ( 0.05 pg/mL). CONCLUSION: Early normal pregnancy is associated with decreased methylation in IL1a, IL1b and ILR2 compared with non-pregnancy levels, which are the same regardless of parity. ILRA and ILR1 are unchanged. Serum IL1b levels, usually non-detectible in healthy controls, are increased in our early pregnancy cohort. Altered methylation in IL1 genes at different gestational time points may result in increased expression as demonstrated by increased IL1b serum levels associated with early pregnancy. 728 Methylation altered in IL1 family of genes in maternal DNA in preeclamptic versus normotensive pregnancy at time of delivery Wendy White, Brian Brost, Jonathan O’Brien, Elizabeth Baldwin, William Watson, Carl Rose, Norman Davies, Zhifu Sun, Stephen Turner, Vesna Garovic Mayo Clinic College of Medicine, Maternal Fetal Medicine, Rochester, MN, Mayo Clinic College of Medicine, Biomedical Statistics and Informatics, Rochester, MN, Mayo Clinic College of Medicine, Nephrology and Hypertension, Rochester, MN OBJECTIVE: The Interleukin 1 family contains Interleukin 1 alpha (IL1a), Interleukin 1 beta (IL1b), IL1 receptor antagonist (IL1RA) and two receptors (IL1R1 and IL1R2). Alterations in the IL1 family have been associated with preeclampsia (PE). IL1a allelic variants have been correlated with PE, but changes in serum levels have not been seen. Serum IL1b levels have been shown to be elevated in PE. Increased expression of IL1RA has been shown in leukocytes from PE women. DNA methylation is an epigenetic mechanism that can control gene expression. We sought to characterize the methylation patterns of 5 IL1 genes in maternal DNA in preeclamptic and normotensive pregnancies at the time of delivery. STUDY DESIGN: The methylation profiles of the IL1 family (10 CpG sites in 5 genes) were characterized in maternal leukocyte DNA from blood in 14 normotensive (NT) women and in 14 PE cases at the time of delivery. PE was diagnosed based on ACOG criteria. All patients were non-smokers, primigravidae and were matched for age and BMI. Genomic DNA was derived from buffy coat, purified, and bisulfite modified then run on the Illumina Methylation Assay platform. Mean methylation levels at each CpG site were compared using a Students t-test. RESULTS: Key CpG sites in the genes for IL1a (0.48 v. 0.44; p 0.01) and IL1b (0.27 v. 0.24; p 0.03; 0.22 v. 0.19; p 0.05) showed increased methylation in PE as compared with NT pregnancies at the time of delivery. Decreased methylation in IL1R1 (0.80 v. 0.83; p 0.03) was seen in PE. No significant difference was seen in methylation patterns in IL1R2 and ILRA. CONCLUSION: Methylation at key CpG sites was altered in the IL1a and IL1b genes (increased) and IL1R1 gene (decreased) in women with PE compared with NT women at the time of delivery. Increased methylation can result in decreased gene expression; our results contrast with the literature in terms of protein levels of IL1b in serum, while IL1a and IL1R1 levels have not been studied in PE. We postulate that increased methylation may represent an endogenous regulatory mechanism to ameliorate the proinflammatory state of PE. www.AJOG.org Academic Issues, Antepartum Fetal Assessment, Genetics, Hypertension, Medical-Surgical-Disease Poster Session V

TIMP 3 and TIMP 4 associated with pregnancy Wendy White, Brian Brost, Jonathan O’Brien, Elizabeth Baldwin, William Watson, Carl Rose, Norman Davies, Todd Stanhope, Zhifu Sun, Stephen Turner, Vesna Garovic Mayo Clinic College of Medicine, Maternal Fetal Medicine, Rochester, MN, Mayo Clinic College of Medicine, Obstetrics and Gynecology, Rochester, MN, Mayo Clinic College of Medicine, Biomedical Statistics and Informatics, Rochester, MN, Mayo Clinic College of Medicine, Nephrology and Hypertension, Rochester, MN OBJECTIVE: Matrix metalloproteinases (MMPs) degrade extracellular matrix; tissue inhibitors for MMPs (TIMPs) regulate this protease activity. Maternal leukocytes at the maternal-fetal interface express MMPs/TIMPs, controlling trophoblast invasion and angiogenesis. Altered expression of MMP 2, 9, 14, and TIMPs 1-4 have been associated with establishment of normal pregnancy and adverse pregnancy outcomes. We sought to characterize differential methylation patterns in pregnancy. STUDY DESIGN: Methylation profiles of MMP 2, 9 and 14 (6 CpG sites in 3 genes) as well as 10 CpG sites in 4 TIMP genes 1-4 were characterized longitudinally in maternal DNA from blood in 14 women at 16 weeks, at delivery and postpartum and compared with 14 nulligravid women. All patients were non-smokers, matched for age and BMI. Genomic DNA was derived from buffy coat, purified, bisulfite modified and run on the Illumina Methylation Assay platform. Mean methylation levels at each CpG site were compared using a Students or paired t-test. RESULTS: Nulligravid and postpartum samples had similar methylation profiles for all genes. MMP 2, TIMP 1 and TIMP 2 were not differentially methylated. MMP 9 and 14 were less methylated in early pregnancy versus postpartum (p 0.0009/p 0.001) and matched nulligravid samples (p 0.02/p 0.009). MMP 9 methylation levels remained lower at delivery compared with postpartum levels (p 0.000006). TIMP 3 was less methylated in early pregnancy versus postpartum (p 0.0014) and nulligravid (p 0.0005). TIMP 4 was less methylated in the pregnant versus postpartum comparison only (p 0.0257). CONCLUSION: Nulligravid and postpartum women have similar methylation profiles for the MMP and TIMP genes tested; any changes associated with normal pregnancy revert back to baseline after delivery. MMP 9, MMP 14, TIMP 3 and TIMP 4 are less methylated in pregnancy compared with non-pregnant controls. Since MMP 9 serum levels are known to increase 15x in pregnancy and elevated MMP 14 primes for angiogenesis, altered methylation may be a mechanism by which expression is controlled.

The proteinuria of preeclampsia is associated with glomerular endotheliosis, and a role for damaged and lost podocytes (glomerular epithelial cells) in the development of proteinuria has received support from studies of patients with glomerular diseases. This study asked whether urinary excretion of viable podocytes, quantified by the expression of podocin and other podocyte-specific proteins, is observed in pregnant women with clinically confirmed preeclampsia. In addition, levels of angiogenic factors implicated in the pathogenesis of preeclampsia were estimated. They included soluble receptor for vascular endothelial growth factor, also called fms-like tyrosine kinase receptor-1 (sFlt-1); placental growth factor (PIGF); and serum soluble endoglin. The cross-sectional study recruited 33 pregnant women with preeclampsia, 11 with HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome, and 23 normotensive pregnant women. All of the 15 preeclamptic women evaluated and also those with HELLP syndrome, but not normotensive control subjects, had podocin-positive cells in their urine. Podocyturia was 100% sensitive and 100% specific for the diagnosis of preeclampsia, and the degree of proteinuria correlated positively with podocyturia. The positive predictive value of urinary podocyte excretion for the presence of preeclampsia was greater than that of sFlt-1, PIGF, or endoglin. Nevertheless, serum levels of sFlt-1 were significantly higher in women with preeclampsia or HELLP syndrome than in normotensive control women, and the same was the case for serum levels of soluble endoglin. Women with preeclampsia or HELLP syndrome had lower serum levels of free PlGF than did normotensive pregnant women. Urine levels of PlGF did not differ from those in normal women. Podocyturia is a very sensitive and specific marker for preeclampsia, and it may contribute to the development of proteinuria in affected women. Further studies may clarify the effects of dysregulated vascular endothelial growth factor on mechanisms regulating podocyte attachment.