Experimental data have revealed the critical role played by 2-methoxy-estradiol, a metabolite of 17β-estradiol, in the pathophysiology of preeclampsia. We used gas chromatography/mass spectrometry to measure a whole panel of hormonal steroids in the plasma from women during the third trimester of their pregnancy.
The population study consists of 24 pregnant patients with different outcomes: normal, or complicated by isolated preeclampsia or by severe preeclampsia with Hemolysis Enzyme Liver Low Platelets (HELLP) syndrome.
17β-estradiol was reduced by 50% in isolated preeclampsia, and by 70% in severe preeclampsia with HELLP syndrome (normal: 8.54 ± 0.9 ng/mL; isolated preeclampsia: 4.65 ± 1.0 ng/mL; severe preeclampsia with HELLP syndrome: 2.64 ± 0.4 ng/mL), as is estrone. Downstream, 2-methoxy-estradiol was decreased only in severe preeclampsia with HELLP syndrome. The concentrations of estrone and 17β-estradiol precursors were comparable between groups, suggesting that placental aromatase is deficient in preeclampsia.
The gradual decrease of estrogen levels with increasing severity of preeclampsia suggests an impairment of placental steroidogenesis.
Preeclampsia (PE) affects from 5-8% of pregnancies, and is characterized by an abrupt onset of hypertension and proteinuria after 20 weeks of gestation. It is a major source of morbidity for both the mother and the fetus, and also a major source of mortality in developing nations. The pathophysiology of the maternal syndrome has been clarified in the last 6 years. Some experimental and clinical research findings support the hypothesis that the symptoms are related to a decrease in free vascular endothelial growth factor (VEGF) in the maternal blood, as a result of capture by sFlt-1, the soluble form of VEGF-receptor 1. Because sFlt-1 promoter is sensitive to hypoxia, a paradigm emerged in which poor placentation leads to aberrant secretion of sFlt-1 and, consequently, to maternal disease. However, further upstream, the mechanisms at work in the primary placental defect are unknown. This is a chicken and egg question: primary overexpression of sFlt-1 could also cause poor placentation, but so far the evidence is insufficient to clarify the situation.
Catechol-O-methyl-transferase (COMT) is an enzyme that plays a critical role in the degradation of catecholamines. It is also responsible for the conversion of 2-hydroxy-estradiol into 2-methoxy-estradiol (2-ME), a metabolite involved in the catabolism of HIF-1α, which is an important mediator in the context of ischemia. In 1988, the activity of COMT was found to be lower in the placentas from preeclamptic women, and in 2008, Kanasaki et al reported that mice deficient for COMT were giving birth in a way that mimics human PE. Although the concentration of catecholamines in wild-type and COMT −/− placentas was not measured, the authors discounted the hypothesis that in the absence of COMT, the PE-like phenotype was related to their catecholamine metabolism for 2 reasons: first, the concentration of catecholamines in plasma was generally reduced during pregnancy and second, inhibiting the degradation of catecholamines by clorgyline did not induce hypertension during pregnancy in wild-type mice. It is noteworthy that the administration of 2-ME rescued the PE-like phenotype in COMT −/− mice, and that in a small number of women (8 preeclamptic and 13 with normal pregnancy), the mean concentration of 2-ME was significantly lower in the context of preeclampsia. The 2-ME, which was initially described as a potent antiangiogenic agent in vitro (in endothelial cells) and in vivo (in a context of tumor angiogenesis), was also shown to facilitate the development of an invasive phenotype in cytotrophoblasts placed in a hypoxic environment. Thus, the effect of 2-ME on angiogenesis could be context-dependent, and in the setting of early pregnancy, 2-ME could promote the establishment of the uteroplacental circulation.
Human 2-ME production involves numerous enzymatic steps in the placenta. Briefly, androgens derived from maternal and fetal adrenal glands are converted into 17β-estradiol (17β-E2) by placental aromatase, which is then metabolized into 2-ME by the sequential actions of the CyP450 and COMT enzymes. Collectively, these facts suggest that placental steroidogenesis could be mechanistically involved in the pathophysiology of human PE. In the current study, we attempt to characterize more fully the steroid profile of preeclamptic pregnancies by using a sensitive, precise, and selective gas chromatography/mass spectrometry (GC/MS) method to screen unconjugated and sulfated steroids. Our main aims were as follows: (1) to establish a steroid profile during the third trimester of pregnancy in the plasma from women delivering in a context of normal pregnancy (NP), isolated preeclampsia (iPE), or severe preeclampsia (sPE) complicated by Hemolysis Enzyme Liver Low Platelets (HELLP) syndrome; and (2) to assess the degree of correlation between the steroids of interest and the 2 angiogenic factors: sFlt-1 and soluble endoglin (sEng). We show in this study that 17β-E2 gradually decreases with the severity of PE, and that estrone (E1) and 2-ME are reduced in severe cases of PE. We also demonstrate for the first time that the production of 2-ME is critically dependent on the concentration of its precursor 17β-E2. Lastly, our data suggest in PE aromatase activity is impaired in the placenta.
Materials and Methods
In a previous study, approved by our local institutional review board, the aim of which was to measure the concentration of the angiogenic factors sFlt-1 and sEng, we had included 82 pregnant women, who had delivered between May 2003 and December 2007 in the Department of Obstetrics of the Hopital de Poissy in France, and who had given their open-ended and informed consent for the storage of plasma and the future analysis of biomarkers of preeclampsia. In the end, 72 of these patients were diagnosed as having preeclampsia, either isolated (n = 49), or associated with HELLP syndrome (n = 23), and 10 had a normal pregnancy outcome. Blood was drawn during their hospital stay for diagnostic purposes from the women with a pathologic outcome, and at a routine consultation before delivery for controls, between 35 and 41 weeks of gestation, to avoid as far as possible any difference in the gestational age. From this cohort, 9 women with iPE, 10 with sPE, and 5 with NP outcome were randomly selected to undergo GC/MS steroid profiling. The following routine clinical and biologic data were available ( Table 1 ): age, number of previous pregnancies, previous history of hypertension, diabetes or nephropathy, smoking status, body mass index, systolic and diastolic blood pressure, platelet count, asparate (ASAT) and alanine aminotransferase (ALAT, peak values), lactate dehydrogenase (LDH, peak value), and proteinuria (peak value), total length of gestation, and the weight of the infant at birth.
|Maternal age, y||32 ± 0.7||33.7 ± 2.2||32.5 ± 2.2|
|Gestational age, d||247.6 ± 11.7||231.9 ± 8.0||203.2 ± 9.2 a|
|Parity||1.8 ± 0.4||1.7 ± 0.3||1.4 ± 0.3|
|Gestity||2 ± 0.3||1.6 ± 0.2||2.2 ± 0.4|
|BMI, kg/m 2||26.0 ± 1.8||24.9 ± 1.8||21.0 ± 0.6 a , d|
|Systolic blood pressure, mm Hg||117.2 ± 2.2||151.9 ± 3.0 a , b||160.4 ± 3.8 c|
|Diastolic blood pressure, mm Hg||70.0 ± 1.5||96.9 ± 3.4 c||101.2 ± 2.7 c|
|Proteinuria, g/L||ND d||1.29 ± 0.4||4.7 ± 0.7 e|
|LDH, UI/L||ND||736 ± 179||954 ± 154|
|ASAT, UI/L||ND||51 ± 12||137 ± 33 f|
|ALAT,UI/L||ND||45 ± 14||141 ± 26 g|
|Platelet count, G/μL||ND||118 ± 16||108 ± 9|
|Birthweight, g||2864 ± 486||1787 ± 227||1317 ± 390 a|
Preeclampsia was defined as hypertension (ie, a systolic blood pressure of at least 140 mm Hg, or a diastolic blood pressure of at least 90 mm Hg, on 2 occasions) and proteinuria (>30 mg/dL on 2 consecutive dipsticks) after 20 weeks of gestation. HELLP syndrome was defined according to the currently used Mississippi classification for HELLP: all these patients had LDH >600 IU/L; a low platelet count (class I: ≤50 G/L; class II ≤100 G/L; class III: ≤150 G/L); and a high level of ASAT or ALAT (class I and II: ≥70 IU/L; class III: ≥40 IU/L).
Soluble endoglin and sFlt-1 were measured as previously described in thawed plasma in duplicate, and in a blind fashion by Enzyme Linked Immunoassay (Quantikine Human Endoglin/CD105; R&D Systems, Abingdon, England), after 1:10 dilution, according to the manufacturer’s instructions.
Measurement of steroid levels by GC/MS
Pregnenolone, progesterone (PROG), and its 3α, 5α/β, 20α/β reduced metabolites, its 16α-, 17α-, and 21-hydroxylated derivatives, glucocorticoids (corticosterone, cortisone and cortisol), androgens (androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone), estrogens (E1, 17β-E2, estriol, 2-ME), and steroid sulfate levels were determined by GC/MS according to the protocol described by Liere et al with minor modifications ( Appendix ). All the validation parameters are shown in Table 2 .