- Email: [email protected]
Increased contents of phospholipids, cholesterol, and lipid peroxides in decidua basalis in women with preeclampsia
Increased contents of phospholipids, cholesterol, and lipid peroxides in decidua basalis in women with preeclampsia Anne Cathrine Staff, MD,a, b Trine Ranheim, MSc, PhD,a Janette Khoury, MD,c and Tore Henriksen, MD, PhDa, c Oslo, Norway OBJECTIVES: Accelerated recovery from preeclampsia has been reported after postpartum curettage. Lipid deposition in decidual spiral arteries (acute atherosis) is a histologic feature of preeclampsia. Increased tissue content of lipids is associated with enhanced formation of lipid peroxides, which are compounds that may induce endothelial dysfunction. We hypothesized that the content of lipids and lipid peroxides is elevated in decidua basalis tissues of women with preeclampsia compared with those of women with uneventful pregnancies. STUDY DESIGN: Decidua basalis tissues were obtained with a vacuum aspiration technique during cesarean delivery from 30 preeclamptic and 34 uneventful pregnancies. Total cholesterol, phospholipids, triglycerides, free fatty acids, and lipid peroxides were quantified. RESULTS: Significantly elevated contents of phospholipids, total cholesterol, and lipid peroxides were found in preeclamptic decidua basalis tissues, whereas the contents of triglycerides and free fatty acids did not differ significantly from those of the control group. CONCLUSIONS: Decidua basalis tissues, with their elevated lipid content, may be a source of lipid compounds that can cause maternal endothelial dysfunction in preeclampsia. (Am J Obstet Gynecol 1999;180:587-92.)
Key words: Decidua basalis, endothelial dysfunction, lipids, lipid peroxides, preeclampsia
Preeclampsia is a major cause of maternal and perinatal morbidity and mortality worldwide. The cause of the disease is still unknown, but there is substantial evidence for dysfunction of the maternal systemic endothelial cells.1 A dominant hypothesis of the pathogenesis of preeclampsia is that compounds from uteroplacental tissues are spread to the maternal circulation, where they may interfere with the endothelium. The endothelial dysfunction that can ensue is considered to be a main cause of the classic clinical features of preeclampsia: hypertension, edema, proteinuria, and activated hemostasis. The observation that postpartum curettage leads to a
From the Institute for Nutrition Research,a the Department of Obstetrics and Gynecology, Aker Hospital,b and the Department of Obstetrics and Gynecology, National Hospital,c University of Oslo. Supported by Throne-Holst Foundation, Aktieselskabet Freia Chokolade Fabriks Medisinske Fond, and the Norwegian Research Council. Presented in part as a poster at the Forty-fifth Annual Meeting of the Society for Gynecologic Investigation, Atlanta, Georgia, March 11-14, 1998. Published in abstract form as Staff AC, Ranheim T, Khoury J, Henriksen T. Increased content of lipids in decidua basalis in patients with preeclampsia [abstract]. J Soc Gynecol Invest 1998;5(Suppl):134A. Received for publication July 9, 1998; revised October 16, 1998; accepted October 29, 1998. Reprint requests: Anne Cathrine Staff, MD, Institute for Nutrition Research, Faculty of Medicine, University of Oslo, PO Box 1046 Blindern, N-0316 Oslo, Norway. Copyright © 1999 by Mosby, Inc. 0002-9378/99 $8.00 + 0 6/1/95477
more rapid normalization of the elevated blood pressure of preeclampsia was reported in 1961 in a nonrandomized study.2 A recent randomized American study concluded that immediate postpartum curettage of women with severe preeclampsia resulted in a faster clinical recovery than that of women who did not undergo curettage.3 Compared with the control group, those who underwent curettage after delivery had a more rapid decrease in the mean arterial pressure, a higher mean urine output, and a faster increase in platelet count. Postpartum curettage involves the removal of decidual tissue, and these studies therfore may imply that decidual components are important in sustaining the preeclampsia syndrome. The spiral arteries in the decidua basalis play an important role in the development of preeclampsia. The restructuring of these arteries into the wider, low-resistance, high-flow vessels seen in normal pregnancies is impaired in women with preeclampsia.4 Defective phenotypic transformation and low invasiveness of the cytotrophoblasts are closely associated with poor remodeling of the spiral arteries.5 Another prominent finding in preeclampsia is histologically visible lipid deposition in the walls of the spiral arteries, a phenomenon known as acute atherosis.6 Among the uteroplacental compounds that may mediate disturbance of the maternal endothelium are lipid products, in particular lipid peroxides.7 Lipid peroxides 587
588 Staff et al
March 1999 Am J Obstet Gynecol
Table I. Clinical characteristics of women included in analyses of various lipid groups in decidua basalis tissues Phospholipids, total cholesterol, triglycerides
Free fatty acids
Preeclampsia Control Statistical Preeclampsia (n = 15) (n = 18) significance (n = 17) Patient age at delivery (y) Body mass index before pregnancy (kg/m2) Body mass index at delivery (kg/m2) Gravidity Parity Pregnancy duration (wk) Neonatal weight (g) Systolic blood pressure at delivery (mm Hg) Diastolic blood pressure at delivery (mm Hg)
Lipid peroxides
Control Statistical Preeclampsia Control (n = 15) significance (n = 16) (n = 17)
Statistical significance
30.5 22.3
32.0 21.2
P = .4 P = .5
28.5 22.8
30.5 20.9
P = .6 P = .2
28.5 23.4
30.5 20.9
P = .6 P = .08
28.1
27.3
P = .3
28.2
26.3
P = .1
28.6
26.3
P = .07
1 0 35.7 2720 170
3 1 38.9 3435 117
P = .005* P = .005* P = .02* P < .001* P < .001*
2 0 34.7 2495 165
2 1 39.1 3260 120
P = .7 P = .3 P = .01* P = .006* P < .001*
2 0 32.5 1963 165
2 1 38.0 3020 120
P = .4 P = .4 P = .02* P < .02* P < .001*
110
78
P < .001*
100
72
P < .001*
103
70
P < .001*
Values are presented as median. *Statistically significant difference between 2 groups at 5% level, according to Mann-Whitney U test.
are produced when free radicals attack polyunsaturated fatty acids and cholesterol in membranes and lipoproteins. Lipid peroxides are highly reactive compounds that may cause cellular dysfunction by several mechanisms, including a direct interaction with cell membranes and an activation of redox-sensitive genes.8 Increased blood levels of lipid peroxidation products in preeclampsia have been reported by several authors.9-11 Placental tissues from women with preeclampsia have been shown to produce more lipid peroxides in vitro than control placentas.12 Although the lipid deposition in spiral arteries has been repeatedly described histologically, it has never been characterized quantitatively or qualitatively by biochemical analysis. Abnormal tissue deposition of lipids, as seen in atherosclerotic arteries, is associated with enhanced production of lipid peroxides.13 On the basis of observations that clinical recovery after curettage is accelerated and observations of the histologic presence of atherotic lipid depositions in the decidual spiral arteries, we hypothesized that preeclamptic decidual tissues could be a source of lipid peroxides and other lipid derivatives. The aim of this study was to establish whether the contents of lipids and lipid peroxides, as measured biochemically, in decidua basalis tissues in preeclamptic pregnancies are elevated with respect to those in control pregnancies. Material and methods Material. Kits for enzymatic determination of triglycerides, phospholipids, and total cholesterol were purchased from bioMérieux (Marcy-l’etoile, France). Triglycerides were determined with the lipase, glycerokinase, glycerol-3-phosphate, and peroxidase method
(Triglycerides Enzymatique PAP 150; bioMérieux). Phospholipids were quantified with the phospholipase D (hydrolyzes choline from lecithin, lysolecithin, and sphingomyelin), choline oxidase, and the peroxidase reaction (Phospholipides enzymatique PAP 150; bioMérieux). Total cholesterol (cholesterol ester and free cholesterol) was measured by means of the cholesterol esterase, the cholesterol oxidase, and the peroxidase reactions (Cholesterol Enzymatique PAP 100; bioMérieux). Deoxyribonucleic acid (DNA) contents in the decidual tissue samples were measured with QIAamp Tisssue kit (QIAGEN, Inc, Valencia, Calif). Nonesterified (free) fatty acid levels were determined with the acyl coenzyme A synthetase and acyl coenzyme A oxidase method (Wako Chemicals USA, Richmond, Va). The kit for determination of lipid peroxides was purchased from Kamiya Biomedical Company (Thousand Oaks, Calif). All organic solutions were of reagent grade. Patient selection. The women selected for this investigation were previously healthy with uncomplicated pregnancies, except for preeclampsia in the study group. In particular, no women with chronic hypertension, renal disease, or diabetes were included. Women in both the preeclamptic and the control group were delivered by the cesarean route. Cesarean delivery was performed in the patients with preeclampsia because vaginal delivery was not considered appropriate in the setting of disease progression, unfavorable cervical ripening, or both. Control subjects were healthy, normotensive women undergoing cesarean delivery for 1 of the following indications: breech presentation, cephalopelvic disproportion, or psychologic reasons. All pregnancies were singletons, and none of the women were in active labor at the time of cesarean delivery.
Staff et al 589
Volume 180, Number 3, Part 1 Am J Obstet Gynecol
Table II. Total cholesterol, phospholipids, and triglycerides analyzed in decidua basalis in preeclamptic pregnancies and in control pregnancies when lipid values are related to wet weight of tissue Preeclampsia (n = 15)
Total cholesterol Phospholipids Triglycerides
Control (n = 18)
Median
95% Confidence interval
Median
95% Confidence interval
Statistical significance
17.4 31.9 0.9
12.8-19.8 25.8-36.7 0.5-1.3
10.6 23.1 0.8
7.8-16.2 17.4-28.0 0.5-1.1
P = .01* P = .01* P = .27
Data are presented in micromoles of lipid per gram wet weight of decidua basalis. *Statistically significant difference between groups at 5% level, according to Mann-Whitney U test.
Table III. Total cholesterol, phospholipids, and triglycerides analyzed in decidua basalis in preeclamptic pregnancies and in control pregnancies when lipid values are related to DNA content of tissue Preeclampsia (n = 14)
Total cholesterol Phospholipids Triglycerides
Control (n = 17)
Median
95% Confidence interval
Median
95% Confidence interval
Statistical significance
19.2 34.9 1.2
11.9-23.8 28.5-46.7 0.4-1.3
10.9 22.3 0.6
6.7-18.1 17.0-31.5 0.6-1.8
P = .03* P = .01* P = .25
Data are presented as nanomoles of lipid per microgram of DNA. *Statistically significant difference between groups at 5% level, according to Mann-Whitney U test.
Preeclampsia was defined as rise in blood pressure after 20 weeks’ gestation to >140/90 mm Hg on ≥2 occasions 6 hours apart in a previously normotensive woman, combined with proteinuria. Proteinuria was defined as a protein dipstick reading of ≥1+ on ≥2 midstream urine samples 6 hours apart. The Korotkoff phase V was used to determine diastolic blood pressure. Pregnancy duration was based on routine ultrasonographic screening between gestational weeks 17 and 20. Decidua basalis samples. We found that the traditional placental bed knife biopsy technique yielded an insufficient amount of decidual tissue. The representative nature of material obtained in this way may also be questionable. We therefore developed a vacuum suction technique through which decidua basalis tissues were obtained by careful vacuum aspiration of the placental bed. After the delivery of the baby, 5 IU oxytocin was given intravenously to the mother and the placenta was located by manual palpation. The placenta was then allowed to separate spontaneously from the uterine wall and was gently removed. Decidual tissues of the placental bed were collected by the suction force directly on a nylon net, which was immediately flushed with 500 mL sterile saline solution to remove blood. A random portion of each sample was taken for histopathologic confirmation of the decidual nature of the tissues. The remaining tissues were snap frozen without delay in liquid nitrogen and stored at –70°C. The decidua basalis samples were collected from 64 cesarean deliveries (a total of 30 preeclamptic gestations and 34 control gestations).
Between 2 and 8 g tissue was obtained per patient. Informed written consent was obtained from each patient. The study protocol was approved by the Regional Committee of Medical Ethics in Norway. Methods. The frozen specimens of decidua basalis tissue were homogenized into powder with a pestle and mortar in liquid nitrogen before analysis. Lipids were extracted by means of the procedure of Folch et al14 with 2:1 chloroform/methanol (vol/vol). The organic phase was dried under a stream of nitrogen and redissolved in the organic solutions described in the following text. To determine triglycerides, phospholipids, and total cholesterol, the lipid extract was redissolved in isopropanol. The kits were therefore applied on an isopropanol solution rather than on plasma. The standard curves were linear when isopropanol was used as a solvent for the standard lipids. All determinations were done on a single aliquot from each patient analyzed in triplicate. The contents of DNA of decidual tissue were measured with QIAamp Tisssue kit in 4 separate tissue samples from each patient. The kit to determine nonesterified (free) fatty acids was modified for tissue analysis by applying the standard reagents on the lipid extract redissolved in ethanol. The standard curves were linear when ethanol was used to dissolve the standard nonesterified free fatty acid solution (oleic acid). Determinations were done on a single aliquot from each patient analyzed in duplicate. The lipid peroxides were determined in 2 separate tis-
590 Staff et al
March 1999 Am J Obstet Gynecol
Table IV. Free fatty acids and lipid peroxides analyzed in decidua basalis in preeclamptic pregnancies and in control pregnancies when lipid values are related to wet weight of tissue Preeclampsia
Free fatty acids Lipid peroxides
Control
Median
95% Confidence interval
No.
Median
95% Confidence interval
1026 7.7
784-1188 5.8-10.9
17 16
1157 4.5
973-1291 3.2-7.4
No. 15 17
Statistical significance P = .2 P = .02*
Data are presented as nanomoles of lipid per gram wet weight of decidua basalis. *Statistically significant difference between groups at 5% level, according to Mann-Whitney U test.
sue samples from each patient, with each sample analyzed in duplicate. For this analysis the lipid extract was redissolved in methanol.15 The kit is based on a colorimetric end point in which hemoglobin catalyzes the reaction of hydroperoxides with a methylene blue derivative, forming an equimolar concentration of methylene blue. The amount of lipid peroxides was calculated with cumene hydroperoxides as standard. A crucial point in this analysis was to avoid any hemoglobin components from the tissue specimens entering into the organic phase. To achieve this the pH in the sodium chloride solution used to create the polar phase was kept between 2 and 2.5. Statistics. The results are presented as the median values and the 95% confidence intervals for the median.16 Statistical analyses, including stepwise regression analysis, were performed by means of the standard programs covered by the Statistical Package for the Social Sciences (version 8.0; SPSS, Chicago, Ill). Differences between groups were tested by nonparametric Mann-Whitney U tests. P < .05 was considered statistically significant. Results The clinical characteristics of the women providing decidual tissues for the lipid analyses are given in Table I. There were no significant differences between the preeclampsia and control groups with respect to age of the women at delivery or with respect to body mass indexes before and at delivery. Median pregnancy duration and baby weight differed statistically significantly between the 2 groups. As expected, because of the inclusion criteria, the blood pressure was statistically significantly elevated in the preeclampsia group with respect to the control group. Tables II and III show the decidual lipid content given per gram of wet weight and per microgram of DNA, respectively. Phospholipids and total cholesterol levels were found to have a statistically significant increase in the preeclampsia group with respect to the control group when either tissue weight or DNA quantity was used as a denominator. The difference in triglyceride levels between the 2 groups was not statistically significant. There was no statistical significant difference in the decidual content of free fatty acids between the preeclamp-
sia and the control groups (Table IV). The powers of the triglyceride and the free fatty acid studies were 84% and 82%, respectively, which means that the number of patients included was acceptable with respect to the risk of type II error.16 A statistically significantly higher content of lipid peroxides was found in the preeclamptic decidua basalis than in that of the control group (Table IV). The effects of pregnancy duration, gravidity, and parity on lipid values were tested with stepwise regression analysis. No statistically significant effect of the pregnancy duration, gravidity, or parity could be demonstrated on the decidual content of either triglycerides, total cholesterol, phospholipids, free fatty acids, or lipid peroxides (all P > .1). This was the case when the preeclampsia and control groups were analyzed both together and separately. The vacuum suction technique proved to be an efficient and simple method for obtaining a sufficient amount of decidual tissue for the lipid analyses. There were no clinical complications related to the vacuum suction. All the decidua basalis specimens were histopathologically characterized by the pathologist as decidual tissue. Comment The vacuum suction technique used resulted in sufficient amounts of tissue for biochemical analysis, in contrast to traditional knife biopsy of the placental bed. Because the placental bed was carefully aspirated, we believe that this technique yields more representative material from the 150 spiral arteries usually present in the decidua basalis than would localized biopsies. Histologically, only decidual tissue was present in all the samples obtained. In this study we quantified the lipid contents of decidua basalis tissues in women with and without preeclampsia. Phospholipids and total cholesterol were increased in preeclamptic decidua basalis tissues. These lipids represent major parts of the cell membranes. The increased content of total cholesterol and phospholipids in the preeclamptic decidual tissues with respect to the control decidual tissues could therefore be caused by an increased number of cells per gram of tissue. However, regardless of whether the content of decidual lipid was
Staff et al 591
Volume 180, Number 3, Part 1 Am J Obstet Gynecol
measured against wet weight of tissue or to quantity of DNA, similar differences between the 2 patient groups were found. Accordingly, the increased lipid content does not seem to be caused by an augmented cellular density in decidua basalis of women with preeclampsia. In contrast to total cholesterol and phospholipids, the levels of triglycerides and nonesterified free fatty acids were similar in the preeclampsia and control groups. Thus the different classes of lipids in preeclamptic decidua tissues were not uniformly increased. It is not known whether the increased content of cholesterol and phospholipids is due to the deposition of plasma lipids or to local synthesis. Deposition of plasma lipoproteins, and not local lipid synthesis, is the main source of the lipids accumulating in atherosclerotic lesions.17 Pregnancy in general, and preeclampsia in particular, is associated with a marked hyperlipidemia.18 It is therefore conceivable, by analogy with atherosclerosis, that deposition of plasma lipids is an important cause of increased decidual lipids in preeclampsia. The preeclamptic hyperlipidemia is characterized by elevated plasma triglycerides and free fatty acids.18, 19 We did not find increased decidual content of these 2 lipid classes in preeclampsia compared with uneventful pregnancies. Triglycerides have a high turnover, however, and they may rapidly be hydrolyzed at the endothelial surface. The low-density lipoprotein subclass of small dense low-density lipoprotein is increased in women with preeclampsia.20 Low-density lipoprotein particles are rich in cholesterol and phospholipids. Small dense low-density lipoprotein could therefore be a source of decidual lipids if deposition of plasma lipids plays a role in inducing lipid accumulation in preeclamptic decidual tissues. So far we have not been able to perform a satisfactory separation of the spiral arteries from the rest of the decidual tissue. On the basis of histologic evidence, however, the large proportion of the decidual lipids would expectedly be localized to the arterial walls.4, 6 We found an elevated level of lipid peroxides in decidua basalis tissues in preeclampsia. When lipid peroxides are present in a tissue, other oxidative derivatives of fatty acids and cholesterol are expected to be present. This is well documented for atherosclerotic lesions, in which both isoprostanes and oxidized sterol compounds are found.21, 22 Increased release of isoprostanes has been reported from placental tissue of preeclamptic pregnancies,23 which indicates that oxidative lipid products other than lipid peroxides were present in placental tissues. Several mechanisms may lead to the increased decidual content of lipid peroxides described in this article. Episodes of localized, intermittent ischemia may occur as a result of the poor transformation of the spiral arteries in preeclampsia. Intermittent ischemia is associated with enhanced formation of free radicals and initiation of lipid peroxidation.24 In the presence of increased tissue
content of lipids, there are more lipid double bonds available for free radical attacks, resulting in potentiated production of lipid peroxides. In the presence of reduced antioxidant capacity, the oxidative stress may further increase. Whether the local antioxidant capacity is altered in preeclamptic decidual tissues is unknown, but there is evidence that plasma antioxidant capacity is reduced in preeclampsia at the end of the pregnancy.25 The observation that postpartum curettage accelerates recovery from preeclampsia indicates that decidual components are important in sustaining preeclamptic disease. We suggest that the decidua basalis could be an important source of compounds, such as oxidative lipid products, that in the systemic maternal circulation could induce endothelial dysfunction. We are grateful for the assistance in tissue sampling of the obstetric staff at Aker University Hospital, in particular, the gynecologists C. Falck, S. Sand, E. Lande, L. Berge, and B. Lorentzen. The technical assistance of K. Høimyr is acknowledged. We also thank Dr B. Halvorsen for valuable discussion and comments on the manuscript. REFERENCES
1. Roberts JM, Redman CW. Pre-eclampsia: more than pregnancyinduced hypertension. Lancet 1993;341:1447-51. 2. Hunter CA, Howard WF, McCormick CO. Amelioration of the hypertension of toxemia by postpartum curettage. Am J Obstet Gynecol 1961;81:884-9. 3. Magann EF, Martin JN, Isaacs JD, Perry KG, Martin RW, Meydrech EF. Immediate postpartum curettage: accelerated recovery from severe preeclampsia. Obstet Gynecol 1993;81:502-6. 4. Brosens IA, Robertson WB, Dixon HG. The role of the spiral arteries in the pathogenesis of preeclampsia. Obstet Gynecol Annu 1972;1:177-91. 5. Zhou Y, Damsky CH, Fisher SJ. Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype: one cause of defective endovascular invasion in this syndrome? J Clin Invest 1997;99:2152-64. 6. Zeek PM, Assali NS. Vascular changes in the decidua associated with eclamptogenic toxemia of pregnancy. Am J Clin Pathol 1950;20:1099-109. 7. Hubel CA, Roberts JM, Taylor RN, Musci TJ, Rodgers GM, McLaughlin MK. Lipid peroxidation in pregnancy: new perspectives on preeclampsia. Am J Obstet Gynecol 1989;161:1025-34. 8. Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996;10:709-20. 9. Wang Y, Walsh SW, Guo J, Zhang J. The imbalance between thromboxane and prostacyclin in preeclampsia is associated with an imbalance between lipid peroxides and vitamin E in maternal blood. Am J Obstet Gynecol 1991;165:1695-700. 10. Ishihara M. Studies on lipoperoxide of normal pregnant women and of patients with toxemia of pregnancy. Clin Chim Acta 1978;84:1-9. 11. Maseki M, Nishigaki I, Hagihara M, Tomoda Y, Yagi K. Lipid peroxide levels and lipid content of serum lipoprotein fractions of pregnant subjects with or without pre-eclampsia. Clin Chim Acta 1981;115:155-61. 12. Walsh SW. Lipid peroxidation in pregnancy. Hypertens Pregnancy 1994;13:1-32. 13. Glavind J, Hartmann S, Clemmesen J, Jessen KE, Dam H. Studies on the role of lipoperoxides in human pathology: the presence of peroxidized lipids in the atherosclerotic aorta. Acta Pathol Microbiol Scand 1951;30:1-6. 14. Folch J, Lees M, Sloane Stanley GH. A simple method for the
592 Staff et al
15.
16. 17. 18.
19.
20.
isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497-509. Yagi K, Kiuchi K, Saito Y, Miike A, Kayahara N, Tatano T, et al. Use of a new methylene blue derivative for determination of lipid peroxides in foods. Biochem Int 1986;12:367-71. Altman DG. Practical statistics for medical research. London: Chapman & Hall; 1991. p. 455-60, 535-7. Smith EB. The relationship between plasma and tissue lipids in human atherosclerosis. Adv Lipid Res 1974;12:1-49. Potter JM, Nestel PJ. The hyperlipidemia of pregnancy in normal and complicated pregnancies. Am J Obstet Gynecol 1979;133:165-70. Lorentzen B, Drevon CA, Endresen MJ, Henriksen T. Fatty acid pattern of esterified and free fatty acids in sera of women with normal and pre-eclamptic pregnancy. Br J Obstet Gynaecol 1995;102:530-7. Sattar N, Bendomir A, Berry C, Shepherd J, Greer IA, Packard CJ. Lipoprotein subfraction concentrations in preeclampsia:
March 1999 Am J Obstet Gynecol
21.
22. 23.
24.
25.
pathogenic parallels to atherosclerosis. Obstet Gynecol 1997;89:403-8. Gniwotta C, Morrow JD, Roberts LJ, Kuhn H. Prostaglandin F2like compounds, F2-isoprostanes, are present in increased amounts in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol 1997;17:3236-41. Brooks CJ, Steel G, Gilbert JD, Harland WA. Lipids of human atheroma. Atherosclerosis 1971;223-37. Walsh SW, Wang Y, Vaughan JE. Placental production of 8-isoprostane is significantly increased in preeclampsia [abstract]. J Soc Gynecol Investig 1997;4(Suppl):96A. Bolli R, Jeroudi MO, Patel BS, Aruoma OI, Halliwell B, Lai E, et al. Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion. Circ Res 1989;65:607-22. Davidge ST, Hubel CA, Brayden RD, Capeless EC, McLaughlin MK. Sera antioxidant activity in uncomplicated and preeclamptic pregnancies. Obstet Gynecol 1992;79:897-901.
Key words: Decidua basalis, endothelial dysfunction, lipids, lipid peroxides, preeclampsia
Preeclampsia is a major cause of maternal and perinatal morbidity and mortality worldwide. The cause of the disease is still unknown, but there is substantial evidence for dysfunction of the maternal systemic endothelial cells.1 A dominant hypothesis of the pathogenesis of preeclampsia is that compounds from uteroplacental tissues are spread to the maternal circulation, where they may interfere with the endothelium. The endothelial dysfunction that can ensue is considered to be a main cause of the classic clinical features of preeclampsia: hypertension, edema, proteinuria, and activated hemostasis. The observation that postpartum curettage leads to a
From the Institute for Nutrition Research,a the Department of Obstetrics and Gynecology, Aker Hospital,b and the Department of Obstetrics and Gynecology, National Hospital,c University of Oslo. Supported by Throne-Holst Foundation, Aktieselskabet Freia Chokolade Fabriks Medisinske Fond, and the Norwegian Research Council. Presented in part as a poster at the Forty-fifth Annual Meeting of the Society for Gynecologic Investigation, Atlanta, Georgia, March 11-14, 1998. Published in abstract form as Staff AC, Ranheim T, Khoury J, Henriksen T. Increased content of lipids in decidua basalis in patients with preeclampsia [abstract]. J Soc Gynecol Invest 1998;5(Suppl):134A. Received for publication July 9, 1998; revised October 16, 1998; accepted October 29, 1998. Reprint requests: Anne Cathrine Staff, MD, Institute for Nutrition Research, Faculty of Medicine, University of Oslo, PO Box 1046 Blindern, N-0316 Oslo, Norway. Copyright © 1999 by Mosby, Inc. 0002-9378/99 $8.00 + 0 6/1/95477
more rapid normalization of the elevated blood pressure of preeclampsia was reported in 1961 in a nonrandomized study.2 A recent randomized American study concluded that immediate postpartum curettage of women with severe preeclampsia resulted in a faster clinical recovery than that of women who did not undergo curettage.3 Compared with the control group, those who underwent curettage after delivery had a more rapid decrease in the mean arterial pressure, a higher mean urine output, and a faster increase in platelet count. Postpartum curettage involves the removal of decidual tissue, and these studies therfore may imply that decidual components are important in sustaining the preeclampsia syndrome. The spiral arteries in the decidua basalis play an important role in the development of preeclampsia. The restructuring of these arteries into the wider, low-resistance, high-flow vessels seen in normal pregnancies is impaired in women with preeclampsia.4 Defective phenotypic transformation and low invasiveness of the cytotrophoblasts are closely associated with poor remodeling of the spiral arteries.5 Another prominent finding in preeclampsia is histologically visible lipid deposition in the walls of the spiral arteries, a phenomenon known as acute atherosis.6 Among the uteroplacental compounds that may mediate disturbance of the maternal endothelium are lipid products, in particular lipid peroxides.7 Lipid peroxides 587
588 Staff et al
March 1999 Am J Obstet Gynecol
Table I. Clinical characteristics of women included in analyses of various lipid groups in decidua basalis tissues Phospholipids, total cholesterol, triglycerides
Free fatty acids
Preeclampsia Control Statistical Preeclampsia (n = 15) (n = 18) significance (n = 17) Patient age at delivery (y) Body mass index before pregnancy (kg/m2) Body mass index at delivery (kg/m2) Gravidity Parity Pregnancy duration (wk) Neonatal weight (g) Systolic blood pressure at delivery (mm Hg) Diastolic blood pressure at delivery (mm Hg)
Lipid peroxides
Control Statistical Preeclampsia Control (n = 15) significance (n = 16) (n = 17)
Statistical significance
30.5 22.3
32.0 21.2
P = .4 P = .5
28.5 22.8
30.5 20.9
P = .6 P = .2
28.5 23.4
30.5 20.9
P = .6 P = .08
28.1
27.3
P = .3
28.2
26.3
P = .1
28.6
26.3
P = .07
1 0 35.7 2720 170
3 1 38.9 3435 117
P = .005* P = .005* P = .02* P < .001* P < .001*
2 0 34.7 2495 165
2 1 39.1 3260 120
P = .7 P = .3 P = .01* P = .006* P < .001*
2 0 32.5 1963 165
2 1 38.0 3020 120
P = .4 P = .4 P = .02* P < .02* P < .001*
110
78
P < .001*
100
72
P < .001*
103
70
P < .001*
Values are presented as median. *Statistically significant difference between 2 groups at 5% level, according to Mann-Whitney U test.
are produced when free radicals attack polyunsaturated fatty acids and cholesterol in membranes and lipoproteins. Lipid peroxides are highly reactive compounds that may cause cellular dysfunction by several mechanisms, including a direct interaction with cell membranes and an activation of redox-sensitive genes.8 Increased blood levels of lipid peroxidation products in preeclampsia have been reported by several authors.9-11 Placental tissues from women with preeclampsia have been shown to produce more lipid peroxides in vitro than control placentas.12 Although the lipid deposition in spiral arteries has been repeatedly described histologically, it has never been characterized quantitatively or qualitatively by biochemical analysis. Abnormal tissue deposition of lipids, as seen in atherosclerotic arteries, is associated with enhanced production of lipid peroxides.13 On the basis of observations that clinical recovery after curettage is accelerated and observations of the histologic presence of atherotic lipid depositions in the decidual spiral arteries, we hypothesized that preeclamptic decidual tissues could be a source of lipid peroxides and other lipid derivatives. The aim of this study was to establish whether the contents of lipids and lipid peroxides, as measured biochemically, in decidua basalis tissues in preeclamptic pregnancies are elevated with respect to those in control pregnancies. Material and methods Material. Kits for enzymatic determination of triglycerides, phospholipids, and total cholesterol were purchased from bioMérieux (Marcy-l’etoile, France). Triglycerides were determined with the lipase, glycerokinase, glycerol-3-phosphate, and peroxidase method
(Triglycerides Enzymatique PAP 150; bioMérieux). Phospholipids were quantified with the phospholipase D (hydrolyzes choline from lecithin, lysolecithin, and sphingomyelin), choline oxidase, and the peroxidase reaction (Phospholipides enzymatique PAP 150; bioMérieux). Total cholesterol (cholesterol ester and free cholesterol) was measured by means of the cholesterol esterase, the cholesterol oxidase, and the peroxidase reactions (Cholesterol Enzymatique PAP 100; bioMérieux). Deoxyribonucleic acid (DNA) contents in the decidual tissue samples were measured with QIAamp Tisssue kit (QIAGEN, Inc, Valencia, Calif). Nonesterified (free) fatty acid levels were determined with the acyl coenzyme A synthetase and acyl coenzyme A oxidase method (Wako Chemicals USA, Richmond, Va). The kit for determination of lipid peroxides was purchased from Kamiya Biomedical Company (Thousand Oaks, Calif). All organic solutions were of reagent grade. Patient selection. The women selected for this investigation were previously healthy with uncomplicated pregnancies, except for preeclampsia in the study group. In particular, no women with chronic hypertension, renal disease, or diabetes were included. Women in both the preeclamptic and the control group were delivered by the cesarean route. Cesarean delivery was performed in the patients with preeclampsia because vaginal delivery was not considered appropriate in the setting of disease progression, unfavorable cervical ripening, or both. Control subjects were healthy, normotensive women undergoing cesarean delivery for 1 of the following indications: breech presentation, cephalopelvic disproportion, or psychologic reasons. All pregnancies were singletons, and none of the women were in active labor at the time of cesarean delivery.
Staff et al 589
Volume 180, Number 3, Part 1 Am J Obstet Gynecol
Table II. Total cholesterol, phospholipids, and triglycerides analyzed in decidua basalis in preeclamptic pregnancies and in control pregnancies when lipid values are related to wet weight of tissue Preeclampsia (n = 15)
Total cholesterol Phospholipids Triglycerides
Control (n = 18)
Median
95% Confidence interval
Median
95% Confidence interval
Statistical significance
17.4 31.9 0.9
12.8-19.8 25.8-36.7 0.5-1.3
10.6 23.1 0.8
7.8-16.2 17.4-28.0 0.5-1.1
P = .01* P = .01* P = .27
Data are presented in micromoles of lipid per gram wet weight of decidua basalis. *Statistically significant difference between groups at 5% level, according to Mann-Whitney U test.
Table III. Total cholesterol, phospholipids, and triglycerides analyzed in decidua basalis in preeclamptic pregnancies and in control pregnancies when lipid values are related to DNA content of tissue Preeclampsia (n = 14)
Total cholesterol Phospholipids Triglycerides
Control (n = 17)
Median
95% Confidence interval
Median
95% Confidence interval
Statistical significance
19.2 34.9 1.2
11.9-23.8 28.5-46.7 0.4-1.3
10.9 22.3 0.6
6.7-18.1 17.0-31.5 0.6-1.8
P = .03* P = .01* P = .25
Data are presented as nanomoles of lipid per microgram of DNA. *Statistically significant difference between groups at 5% level, according to Mann-Whitney U test.
Preeclampsia was defined as rise in blood pressure after 20 weeks’ gestation to >140/90 mm Hg on ≥2 occasions 6 hours apart in a previously normotensive woman, combined with proteinuria. Proteinuria was defined as a protein dipstick reading of ≥1+ on ≥2 midstream urine samples 6 hours apart. The Korotkoff phase V was used to determine diastolic blood pressure. Pregnancy duration was based on routine ultrasonographic screening between gestational weeks 17 and 20. Decidua basalis samples. We found that the traditional placental bed knife biopsy technique yielded an insufficient amount of decidual tissue. The representative nature of material obtained in this way may also be questionable. We therefore developed a vacuum suction technique through which decidua basalis tissues were obtained by careful vacuum aspiration of the placental bed. After the delivery of the baby, 5 IU oxytocin was given intravenously to the mother and the placenta was located by manual palpation. The placenta was then allowed to separate spontaneously from the uterine wall and was gently removed. Decidual tissues of the placental bed were collected by the suction force directly on a nylon net, which was immediately flushed with 500 mL sterile saline solution to remove blood. A random portion of each sample was taken for histopathologic confirmation of the decidual nature of the tissues. The remaining tissues were snap frozen without delay in liquid nitrogen and stored at –70°C. The decidua basalis samples were collected from 64 cesarean deliveries (a total of 30 preeclamptic gestations and 34 control gestations).
Between 2 and 8 g tissue was obtained per patient. Informed written consent was obtained from each patient. The study protocol was approved by the Regional Committee of Medical Ethics in Norway. Methods. The frozen specimens of decidua basalis tissue were homogenized into powder with a pestle and mortar in liquid nitrogen before analysis. Lipids were extracted by means of the procedure of Folch et al14 with 2:1 chloroform/methanol (vol/vol). The organic phase was dried under a stream of nitrogen and redissolved in the organic solutions described in the following text. To determine triglycerides, phospholipids, and total cholesterol, the lipid extract was redissolved in isopropanol. The kits were therefore applied on an isopropanol solution rather than on plasma. The standard curves were linear when isopropanol was used as a solvent for the standard lipids. All determinations were done on a single aliquot from each patient analyzed in triplicate. The contents of DNA of decidual tissue were measured with QIAamp Tisssue kit in 4 separate tissue samples from each patient. The kit to determine nonesterified (free) fatty acids was modified for tissue analysis by applying the standard reagents on the lipid extract redissolved in ethanol. The standard curves were linear when ethanol was used to dissolve the standard nonesterified free fatty acid solution (oleic acid). Determinations were done on a single aliquot from each patient analyzed in duplicate. The lipid peroxides were determined in 2 separate tis-
590 Staff et al
March 1999 Am J Obstet Gynecol
Table IV. Free fatty acids and lipid peroxides analyzed in decidua basalis in preeclamptic pregnancies and in control pregnancies when lipid values are related to wet weight of tissue Preeclampsia
Free fatty acids Lipid peroxides
Control
Median
95% Confidence interval
No.
Median
95% Confidence interval
1026 7.7
784-1188 5.8-10.9
17 16
1157 4.5
973-1291 3.2-7.4
No. 15 17
Statistical significance P = .2 P = .02*
Data are presented as nanomoles of lipid per gram wet weight of decidua basalis. *Statistically significant difference between groups at 5% level, according to Mann-Whitney U test.
sue samples from each patient, with each sample analyzed in duplicate. For this analysis the lipid extract was redissolved in methanol.15 The kit is based on a colorimetric end point in which hemoglobin catalyzes the reaction of hydroperoxides with a methylene blue derivative, forming an equimolar concentration of methylene blue. The amount of lipid peroxides was calculated with cumene hydroperoxides as standard. A crucial point in this analysis was to avoid any hemoglobin components from the tissue specimens entering into the organic phase. To achieve this the pH in the sodium chloride solution used to create the polar phase was kept between 2 and 2.5. Statistics. The results are presented as the median values and the 95% confidence intervals for the median.16 Statistical analyses, including stepwise regression analysis, were performed by means of the standard programs covered by the Statistical Package for the Social Sciences (version 8.0; SPSS, Chicago, Ill). Differences between groups were tested by nonparametric Mann-Whitney U tests. P < .05 was considered statistically significant. Results The clinical characteristics of the women providing decidual tissues for the lipid analyses are given in Table I. There were no significant differences between the preeclampsia and control groups with respect to age of the women at delivery or with respect to body mass indexes before and at delivery. Median pregnancy duration and baby weight differed statistically significantly between the 2 groups. As expected, because of the inclusion criteria, the blood pressure was statistically significantly elevated in the preeclampsia group with respect to the control group. Tables II and III show the decidual lipid content given per gram of wet weight and per microgram of DNA, respectively. Phospholipids and total cholesterol levels were found to have a statistically significant increase in the preeclampsia group with respect to the control group when either tissue weight or DNA quantity was used as a denominator. The difference in triglyceride levels between the 2 groups was not statistically significant. There was no statistical significant difference in the decidual content of free fatty acids between the preeclamp-
sia and the control groups (Table IV). The powers of the triglyceride and the free fatty acid studies were 84% and 82%, respectively, which means that the number of patients included was acceptable with respect to the risk of type II error.16 A statistically significantly higher content of lipid peroxides was found in the preeclamptic decidua basalis than in that of the control group (Table IV). The effects of pregnancy duration, gravidity, and parity on lipid values were tested with stepwise regression analysis. No statistically significant effect of the pregnancy duration, gravidity, or parity could be demonstrated on the decidual content of either triglycerides, total cholesterol, phospholipids, free fatty acids, or lipid peroxides (all P > .1). This was the case when the preeclampsia and control groups were analyzed both together and separately. The vacuum suction technique proved to be an efficient and simple method for obtaining a sufficient amount of decidual tissue for the lipid analyses. There were no clinical complications related to the vacuum suction. All the decidua basalis specimens were histopathologically characterized by the pathologist as decidual tissue. Comment The vacuum suction technique used resulted in sufficient amounts of tissue for biochemical analysis, in contrast to traditional knife biopsy of the placental bed. Because the placental bed was carefully aspirated, we believe that this technique yields more representative material from the 150 spiral arteries usually present in the decidua basalis than would localized biopsies. Histologically, only decidual tissue was present in all the samples obtained. In this study we quantified the lipid contents of decidua basalis tissues in women with and without preeclampsia. Phospholipids and total cholesterol were increased in preeclamptic decidua basalis tissues. These lipids represent major parts of the cell membranes. The increased content of total cholesterol and phospholipids in the preeclamptic decidual tissues with respect to the control decidual tissues could therefore be caused by an increased number of cells per gram of tissue. However, regardless of whether the content of decidual lipid was
Staff et al 591
Volume 180, Number 3, Part 1 Am J Obstet Gynecol
measured against wet weight of tissue or to quantity of DNA, similar differences between the 2 patient groups were found. Accordingly, the increased lipid content does not seem to be caused by an augmented cellular density in decidua basalis of women with preeclampsia. In contrast to total cholesterol and phospholipids, the levels of triglycerides and nonesterified free fatty acids were similar in the preeclampsia and control groups. Thus the different classes of lipids in preeclamptic decidua tissues were not uniformly increased. It is not known whether the increased content of cholesterol and phospholipids is due to the deposition of plasma lipids or to local synthesis. Deposition of plasma lipoproteins, and not local lipid synthesis, is the main source of the lipids accumulating in atherosclerotic lesions.17 Pregnancy in general, and preeclampsia in particular, is associated with a marked hyperlipidemia.18 It is therefore conceivable, by analogy with atherosclerosis, that deposition of plasma lipids is an important cause of increased decidual lipids in preeclampsia. The preeclamptic hyperlipidemia is characterized by elevated plasma triglycerides and free fatty acids.18, 19 We did not find increased decidual content of these 2 lipid classes in preeclampsia compared with uneventful pregnancies. Triglycerides have a high turnover, however, and they may rapidly be hydrolyzed at the endothelial surface. The low-density lipoprotein subclass of small dense low-density lipoprotein is increased in women with preeclampsia.20 Low-density lipoprotein particles are rich in cholesterol and phospholipids. Small dense low-density lipoprotein could therefore be a source of decidual lipids if deposition of plasma lipids plays a role in inducing lipid accumulation in preeclamptic decidual tissues. So far we have not been able to perform a satisfactory separation of the spiral arteries from the rest of the decidual tissue. On the basis of histologic evidence, however, the large proportion of the decidual lipids would expectedly be localized to the arterial walls.4, 6 We found an elevated level of lipid peroxides in decidua basalis tissues in preeclampsia. When lipid peroxides are present in a tissue, other oxidative derivatives of fatty acids and cholesterol are expected to be present. This is well documented for atherosclerotic lesions, in which both isoprostanes and oxidized sterol compounds are found.21, 22 Increased release of isoprostanes has been reported from placental tissue of preeclamptic pregnancies,23 which indicates that oxidative lipid products other than lipid peroxides were present in placental tissues. Several mechanisms may lead to the increased decidual content of lipid peroxides described in this article. Episodes of localized, intermittent ischemia may occur as a result of the poor transformation of the spiral arteries in preeclampsia. Intermittent ischemia is associated with enhanced formation of free radicals and initiation of lipid peroxidation.24 In the presence of increased tissue
content of lipids, there are more lipid double bonds available for free radical attacks, resulting in potentiated production of lipid peroxides. In the presence of reduced antioxidant capacity, the oxidative stress may further increase. Whether the local antioxidant capacity is altered in preeclamptic decidual tissues is unknown, but there is evidence that plasma antioxidant capacity is reduced in preeclampsia at the end of the pregnancy.25 The observation that postpartum curettage accelerates recovery from preeclampsia indicates that decidual components are important in sustaining preeclamptic disease. We suggest that the decidua basalis could be an important source of compounds, such as oxidative lipid products, that in the systemic maternal circulation could induce endothelial dysfunction. We are grateful for the assistance in tissue sampling of the obstetric staff at Aker University Hospital, in particular, the gynecologists C. Falck, S. Sand, E. Lande, L. Berge, and B. Lorentzen. The technical assistance of K. Høimyr is acknowledged. We also thank Dr B. Halvorsen for valuable discussion and comments on the manuscript. REFERENCES
1. Roberts JM, Redman CW. Pre-eclampsia: more than pregnancyinduced hypertension. Lancet 1993;341:1447-51. 2. Hunter CA, Howard WF, McCormick CO. Amelioration of the hypertension of toxemia by postpartum curettage. Am J Obstet Gynecol 1961;81:884-9. 3. Magann EF, Martin JN, Isaacs JD, Perry KG, Martin RW, Meydrech EF. Immediate postpartum curettage: accelerated recovery from severe preeclampsia. Obstet Gynecol 1993;81:502-6. 4. Brosens IA, Robertson WB, Dixon HG. The role of the spiral arteries in the pathogenesis of preeclampsia. Obstet Gynecol Annu 1972;1:177-91. 5. Zhou Y, Damsky CH, Fisher SJ. Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype: one cause of defective endovascular invasion in this syndrome? J Clin Invest 1997;99:2152-64. 6. Zeek PM, Assali NS. Vascular changes in the decidua associated with eclamptogenic toxemia of pregnancy. Am J Clin Pathol 1950;20:1099-109. 7. Hubel CA, Roberts JM, Taylor RN, Musci TJ, Rodgers GM, McLaughlin MK. Lipid peroxidation in pregnancy: new perspectives on preeclampsia. Am J Obstet Gynecol 1989;161:1025-34. 8. Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996;10:709-20. 9. Wang Y, Walsh SW, Guo J, Zhang J. The imbalance between thromboxane and prostacyclin in preeclampsia is associated with an imbalance between lipid peroxides and vitamin E in maternal blood. Am J Obstet Gynecol 1991;165:1695-700. 10. Ishihara M. Studies on lipoperoxide of normal pregnant women and of patients with toxemia of pregnancy. Clin Chim Acta 1978;84:1-9. 11. Maseki M, Nishigaki I, Hagihara M, Tomoda Y, Yagi K. Lipid peroxide levels and lipid content of serum lipoprotein fractions of pregnant subjects with or without pre-eclampsia. Clin Chim Acta 1981;115:155-61. 12. Walsh SW. Lipid peroxidation in pregnancy. Hypertens Pregnancy 1994;13:1-32. 13. Glavind J, Hartmann S, Clemmesen J, Jessen KE, Dam H. Studies on the role of lipoperoxides in human pathology: the presence of peroxidized lipids in the atherosclerotic aorta. Acta Pathol Microbiol Scand 1951;30:1-6. 14. Folch J, Lees M, Sloane Stanley GH. A simple method for the
592 Staff et al
15.
16. 17. 18.
19.
20.
isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497-509. Yagi K, Kiuchi K, Saito Y, Miike A, Kayahara N, Tatano T, et al. Use of a new methylene blue derivative for determination of lipid peroxides in foods. Biochem Int 1986;12:367-71. Altman DG. Practical statistics for medical research. London: Chapman & Hall; 1991. p. 455-60, 535-7. Smith EB. The relationship between plasma and tissue lipids in human atherosclerosis. Adv Lipid Res 1974;12:1-49. Potter JM, Nestel PJ. The hyperlipidemia of pregnancy in normal and complicated pregnancies. Am J Obstet Gynecol 1979;133:165-70. Lorentzen B, Drevon CA, Endresen MJ, Henriksen T. Fatty acid pattern of esterified and free fatty acids in sera of women with normal and pre-eclamptic pregnancy. Br J Obstet Gynaecol 1995;102:530-7. Sattar N, Bendomir A, Berry C, Shepherd J, Greer IA, Packard CJ. Lipoprotein subfraction concentrations in preeclampsia:
March 1999 Am J Obstet Gynecol
21.
22. 23.
24.
25.
pathogenic parallels to atherosclerosis. Obstet Gynecol 1997;89:403-8. Gniwotta C, Morrow JD, Roberts LJ, Kuhn H. Prostaglandin F2like compounds, F2-isoprostanes, are present in increased amounts in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol 1997;17:3236-41. Brooks CJ, Steel G, Gilbert JD, Harland WA. Lipids of human atheroma. Atherosclerosis 1971;223-37. Walsh SW, Wang Y, Vaughan JE. Placental production of 8-isoprostane is significantly increased in preeclampsia [abstract]. J Soc Gynecol Investig 1997;4(Suppl):96A. Bolli R, Jeroudi MO, Patel BS, Aruoma OI, Halliwell B, Lai E, et al. Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion. Circ Res 1989;65:607-22. Davidge ST, Hubel CA, Brayden RD, Capeless EC, McLaughlin MK. Sera antioxidant activity in uncomplicated and preeclamptic pregnancies. Obstet Gynecol 1992;79:897-901.