Med. J. Cairo Univ., Vol. 78, No. 2, September: 117-121, 2010 www.medicaljournalofcairouniversity.

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Maternal Serum Soluble Endoglin in Patients with Pre-Eclampsia and Gestational Hypertension and its Relation to Doppler Study of the Fetomaternal Circulation
NAWARA M. HASHEESH, M.D.; MOHAMED WALY, M.D.; MARWA GOUDA, M.Sc.* and NAGWA EL TAWEEL, M.D.**
The Departments of Obstetrics & Gynaecology, Cairo University & Boulak Hospital* and Clinical Pathology**, Cairo University.

Abstract
Bachground: Inadequate trophoblastic invasion and spiral artery remodelling leading to poor placental perfusion and hypoxia are believed to underlie preeclampsia. Recent studies implicate increased circulating endoglin (an anti-angiogenic factor produced due to placental hypoxia) as a contributer to the pathogenesis of preeclampsia. Objective: The objective of this study is to determine whether soluble Endoglin (sEng) concentrations are altered in pregnancies complicated with different hypertensive disorders of pregnancy as gestational hypertension and preeclampsia and to address its relation with Doppler study of the fetomaternal blood flow. Patients and Methods: We evaluated sEng level in sera of 30 patients with gestational hypertension and 30 patients with preeclampsia after diagnosis of the disease and compared them with 30 normal controls matched for gestational age, parity and BMI and correlated the results with fetomaternal blood flow as assessed by Doppler study. Results: There was significant association between blood pressure augmentation in different hypertensive diseases of pregnancy and soluble endoglin level in serum (9.2ng/ml in controls, 12.1ng/ml in gestational hypertension, 15.3ng/ml in pre-eclampsia). Also patients with abnormal umbilical artery Doppler velocimetry had the highest median serum concentration of sEng. Conclusion: Altered anti-angiogenic factor might be involved in the pathogenesis of the different hypertensive diseases of pregnancy. sEng had a good discriminatory ability between cases of preeclampsia, gestational hypertension and controls. Our data suggest that sEng level correlates positively with vascular dysfunction as evidenced by increased resistance to umbilical blood flow assessed by Doppler study with high sEng level. Our results are consistent with the hypothesis that angiogenic balance plays a role in maternal breast cancer risk reduction associated with blood pressure increases in pregnancy.

Key Words: Endoglin Geslational hypertension Preeclampsia Doppler.

Introduction PREECLAMPSIA is a major cause of maternal perinatal morbidity and mortality. Preeclampsia (PE) occurs in 2-5% of pregnancies in the occident but it complicates up to 10% of pregnancies in the developing countries [1] . The treatment of PE has not changed significantly over the last 50 years because the etiology is still unclear however the central role played by the placenta in its pathology is a certainty. One of the commonly accepted hypothesis is that PE develops as a sequence of some kind of immune maladaptation between the mother and the fetus during the first weeks of pregnancy leading to impaired tissue and arterial invasion by trophoblastic cells resulting in worsened placental perfusion and chronic hypoxia expected to trigger increased placental apoptosis and increased shedding of placentally produced debris and the over-expression of pro-inflammatory, anti-angiogenic and angiogenic factors leading to systemic endothelial damage and an exaggerated inflammatory response as the maternal vascular and immune system can not handle any longer the products of placental oxidative stress [2,3] . Based on these pathophysiological observations, several biochemical markers have been investigated which could provide a non-invasive and affordable method for prediction of this life-threatening disorder and thus providing special medical care for these high risk population. A major focus of research has recently been the identification of placental factors showing abnormal expression in PE

Correspondence to: Dr. Nawara M. Hasheesh, The Department of Obstetrics & Gynaecology, Cairo University.

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Maternal Serum Soluble Endoglin in Patients with Pre-Eclampsia

patients and the assessment of their potential use in early prediction and detection of the disease, one of them is soluble endoglin. Endoglin (Eng) is a co-receptor for transforming growth factor (TGF) that is highly expressed on cellular membranes of the vascular endothelium and on the syncytiotrophoblast. It functions as a modulator of TGF signaling and is involved in angiogenesis and the regulation of the vascular tone. Endoglin is a pro-angiogenic factor that protects endothelial cells under hypoxia and regulate NO (nitric oxide) dependent vasodilatation [4] . On the other hand, soluble Eng (sEng) is an antiangiogenic protein. It consists of the extra cellular part of the molecule that may be produced through the proteo-cleavage of the placental membranebound form. It decreases endothelial nitric oxide by inhibiting TGF signaling and this leads to endothelial dysfunction. In vitro, sEng acts as a negative regulator of angiogenesis by competitive interaction with TGF, thereby impairing capillary formation by endothelial cells. Furthermore, it induces high arterial pressure and vascular permeability in pregnant rats in which the protein was over-expressed. Very interestingly, the introduction of sEng in the pregnant animals induced renal, placental and hepatic changes reminiscent of the HELLP syndrome [5] . Soluble endoglin is present in substantial excess in PE patients compared to normotensive controls however whether this factor is specific for PE or it is also increased with other hypertensive diseases of pregnancy such as gestational or chronic hypertension is the aim of future studies which are needed to clarify this issue. Soluble endoglin could be used as a biochemical marker for prediction of PE in the second trimester, as it is usually increased 10-12 weeks before the clinical symptoms of the disease manifest itself [6,7] . The concentration of sEng appear to increase with the severity of PE especially in PE complicated by HELLP syndrome and IUGR. However, prediction of cut-off value for PE and another for gestational hypertension was not attempted due to the non-consistent results of studies assessing sEng level. Some of these studies showed variable sEng level in pregnancies complicated by PE, different factors affecting sEng level either related to the race or the BMI are still investigated so this point is still a point of discussion and needs further studies. Exploring this biochemical marker potentials are not only restricted to the prediction of the

disease but also to suggested potential therapies as PE is characterized by widespread endothelial damage attributed to two anti-angiogenic factors; VEGFR (vascular endothelial growth factor receptor) and sEng as the co-adminstration of these two factors to pregnant rats elicits severe PE-like symptoms, so these two anti-angiogenic factors are quite possibly the "final common pathway" responsible for the accompanying signs of hypertension and proteinuria as these anti-angiogenic factors are increased dramatically prior to the clinical onset of PE whose symptoms can be reversed by VEGF administration to animal models [8] . Haeme oxygenase-1 (HO-1), an anti-inflammatory enzyme and its metabolite carbonmonoxide (CO) exert protective effects in several organs against oxidative-stimuli. Ahmed and Cudmore 2009, [8] showed that HO-1 pathway inhibit VEGFR and sEng in cultured cells and human placental tissue explants. Both CO and NO (nitric oxide) promote vascular homeostasis and vasodilation. This findings provide compelling evidence for a protective role of HO-1 in pregnancy and identify it as a target for the treatment of PE. Any agent that is known to up-regulate HO-1 such as statins may have potential as a therapy. Aim of the work: Investigating whether maternal circulating levels of the anti-angiogenic factor soluble endoglin in patients with pre-eclampsia or gestational hypertension in the third trimester is higher than its levels in healthy pregnant females of the same gestational age and correlating the sEng level with Doppler study of the fetomaternal circulation. Patients and Methods Detection of maternal circulating levels of soluble endoglin in the third trimester in 30 pregnant women having pre-eclampsia and 30 pregnant women having gestational hypertension and comparing these levels with those detected in 30 healthy pregnant women matched for gestational age, parity and BMI without any medical disorder as controls. Patients with preeclampsia had new onset hypertension (systolic and diastolic blood pressure of 140 and 90mm Hg, respectively, on two occasions, at least 6 hours apart) and proteinuria (protein of >_ 2+), that develop after 20 weeks of gestation in previously normotensive women. Several other symptoms dependent on the systemic involvement, such as oedema, headache, vomiting, blurring of vision, epigastric pain, disturbance of hemostasis, renal or liver function

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disturbances, and the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet counts) were recorded also if present. Patients with gestational hypertension had new onset hypertension (systolic and diastolic blood pressure of 140 and 90mm Hg, respectively, on two occasions, at least 6 hours apart) without proteinuria that develop after 20 weeks of gestation in previously normotensive women. Intra-uterine growth retardation:Any fetus that fails to reach its full growth potential i.e.: Less than 10 th percentile weight for gestational age with abnormal Doppler study of umbilical blood flow. Information regarding the current pregnancy including age, medical and obstetric history and any anti-hypertensive treatment taken were recorded. Exclusion criteria: 1- Women with prior medical disorders as vascular or renal disease. 2- Women with diabetes mellitus or gestational diabetes. 3- Women with twin pregnancy. 4- Women with cancerous lesions. 5- Women who smoke during pregnancy. 6- Women pregnant with fetuses with structural or chromosomal anomalies. Patients were collected from Kasr El-Ainey hospital and Boulak hospital in the period from January 2010 to June 2010 after explaining the procedure and taking informed consent from all participants. Blood samples were collected and the serum was separated and stored at 80 degrees in Clinical pathology department in Kasr El-Ainey hospital and analysed for soluble endoglin level. sEng was measured using an enzyme linked immunosorbent assay (R&D systems, Minneapolis, USA). All the assays were conducted in duplicate and the mean value of the duplicate samples was reported. Minimal detectable levels in the assays was 0.007ng/ml. Results were tabulated to compare soluble endoglin level in pre-eclamptic patients and those with gestational hypertension and healthy pregnant females and this was the primary outcome. Doppler study of the umbilical flow was compared in pre-eclamptic patients and those with gestational hypertension and healthy pregnant females and correlated with sEng level and this was the secondary outcome.

Fetal outcome regarding neonatal birth weight, placental abruption, apgar score and admission to neonatal ICU were recorded. Statistical analysis: Data were statistically described in terms of range, mean standard deviation ( SD), frequencies (number of cases) and percentages when appropriate. Comparison of quantitative variables between the study groups was done using one way analysis of variance (ANOVA) test with posthoc multiple 2-group comparisons. For comparing categorical data, Chi square (X 2 ) test was performed. Exact test was used instead when the expected frequency is less than 5. A probability value ( p -value) less than 0.05 was considered statistically significant. All statistical calculations were done using computer programs Microsoft Excel 2003 (Microsoft corporation, NY, USA) and SPSS (Statistical package for the social science; SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows. Results
Table (1) Preeclamptic group Age Parity Gestational age Systolic BP Diastolic BP Anti-HTN ttt 26.4 6.8 1.17 1.2 36.6 2 167.5 24.7 108.8 11.4 Hypertensive group 28 5.5 1.1 1.2 37 2.3 161.1 15 105.3 8.8 20% Control group 27.2 6.2 1.4 1.4 37.2 1.2 p value 0.638 0.600 0.060

110.6 11.7 0.000 69.2 14.1 0.000

p -value <0.05 is significant.

Table (2) Preeclamptic group Headache Vomiting Blurring of vision Epigastric pain Oedema Eclamptic fits 60% 23.3% 43% 16.6% 56.7% 10% Table (3) HyperPreeclamptic tensive group group EFW 2.6 0.5 Oligohydramnios 16.6% Placental abruption 3.3% Umbilical artery RI 0.7 0.99 IUGR 16.6% 2.8 0.5 6.7% Control group 3.3 0.3 p value Hypertensive group 56.6% 20% 26.6% 6.7% 43.3% Control group

0.000 0.000 0.000 0.64 0.1 0.53 0.05 0.000 6.7% 0.000

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Table (4) Preeclamptic group Hb Platelets Liver enz (ALT) Creatinine Prothrombin Conc. Blood sugar Endoglin 10.2 1 223.3 20.2 24.4 3.9 0.71 0.18 85.3 4.1 84.1 9.4 15.3 1.4 Hypertensive group 10.1 1.2 224.8 17.6 19.94.5 0.72 0.11 84.6 5.4 88.4 9.1 12.1 0.7 Control group

Maternal Serum Soluble Endoglin in Patients with Pre-Eclampsia

p value 0.233 0.000 0.000 0.930 0.000 0.001 0.000

10.5 0.6 242 10.4 14.1 2.9 0.71 0.10 98.3 2.3 83.2 8.3 9.2 1.5

Table (5) HyperPrep Control eclamptic tensive group value group group Mode of delivery: CS Neonatal birth weight Apgar score: 1 min 5 min 13 2.8 0.5 5.3 0.9 7.2 1.1 13 2.9 0.5 5.2 1.1 7 1.5 9 3.4 0.3 6 0.0 8 0.0 0.000 0.002 0.003

studies comparing PIH patients with controls and finding significant difference between them but the mean value of sEng in PIH patients in our study was lower (15.3ng/ml) compared with levels ranging from 20-30ng/ml in Levine study, [10] and Antonio de Vivo study, [11] but this could be attributed to many differences mainly racial as their control was already higher than ours 13-14ng/ml Vs 9.2ng/ml in our study. Also the basic characteristics of the population studied was different; the BMI in our study was >30 compared to a lower BMI in their studies ranging from 23 to 25. Obesity is known to decrease mean sEng level [12] . PE is liable to develop at lower sEng levels in obese patients [13] . Also our cohort consisted mainly of late-onset PE while in other studies, the ratio of early-onset PE was higher and it is well documented that sEng level is higher in early-onset than lateonset PE [14] . Not only can sEng help in the diagnosis of PE but also in its prediction as the study done by Lim and Kim [9] has illustrated that sEng increase consequetively during the pregnancy period in patients destined to develop PE and could be used as a reliable predictor of PE. Levine study [10] also revealed that sEng release from the placenta in the circulation is markedly increased 3 months before PE develops earlier than other angiogenic factors that is why sEng is the most widely studied antiangiogenic factor in the literature and further studies are even expected to clarify the clinical role of this biomarker. A major focus of research has recently been the identification of the altered level of this factor in different ethnic groups and with the different basic characteristics of the population studied such as BMI, insulin resistance and special habits as smoking and intake of medications that could affect the serum level of sEng [5] . The availability of such marker for prediction of PE could provide a rationale for potential future prophylactic and therapeutic interventions for PE. Any treatment achieving even a modest prolongation of pregnancy or amelioration of the condition could have a significant beneficial health impact worldwide. Any agent that could stabilize the negative regulation of sEng for angiogenesis and promote vascular homeostasis and vasodilation such as statins may have potential as a therapy [8] . In addition, the angiogenic profile of PE may provide insight into the underlying mechanism involved in the association of PE with reduced risk of breast cancer. Troisi, [15] explained this association by the relatively anti-angiogenic profile found in PE. Our findings may have implication regarding explaining another similar phenomenon that women

Discussion Studying the angiogenic profile in patients with various hypertensive disorders in pregnancy could be important in understanding the mechanism of blood pressure (BP) augmentation with pregnancy and the development of pre-eclampsia (PE) and whether BP elevation in gestational hypertension (HTN) is associated with alteration in angiogenic profile which could help understanding the pathogenesis of gest. HTN and whether it is a separate entity from PE or a mild version of the disease. The results of our study show a significant elevation of sEng with gest. HTN that is significantly milder than its elevation in PE which indicate that gest. HTN is a mild form of PE and not a separate disease. sEng could also be a sensitive serum biomarker for PE in conjugation with urine protein analysis as the latter is sometimes intermittent and PE may present without signs and symptoms so the use of this biomarker could increase the diagnostic accuracy and prevent the delay in management of PE by differentiating it from other hypertensive diseases of pregnancy as the level of sEng varies between them. In our study, we notified that sEng has a significantly higher mean level in PIH than gest. HTN (15.3ng/ml Vs 12.1ng/ml) and its mean level is significantly higher in gest HTN than controls (12.1ng/ml Vs 9.2ng/ml). This agrees with other

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2- SOLEYMANLOU N., JURISICA I. and NEVO O.: Molecular evidence of placental hypoxia in preeclampsia. J. Clin. Endocrinol. Metab., 90: 4299-4308, 2005. 3- ROBERTS J.M. and HUBEL C.A.: The two stage model of preeclampsia: Variations on the theme. Placenta, 30: S32-S37, 2009. 4- TOPORSIAN M., GROS R., KABIR M.G. and VERA S.: A role for endoglin in coupling eNOS activity and regulating vascular tone revealed in hereditary hemorrhagic telangiectasia. Circ. Res., 96: 684-692, 2005. 5- VENKATESHA S., TOPORSIAN M., LAM C., HANAI J. and MAMMOTO T.: Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat. Med., 12: 642649, 2006. 6- RANA S., KARUMANCHI S. and LEVINE R.J.: Sequential changes in antiangiogenic factors in early pregnancy and risk of developing preeclampsia. Hypertension, 50: 137-42, 2007. 7- ROBINSON C.J. and JOHNSON D.D.: Soluble endoglin as a second-trimester marker for preeclampsia. Am. J. Obstet. Gynecol., 197: 174-175, 2007. 8- AHMED A. and CUDMORE M.J.: Can the biology of VEGF and haem oxygenases help solve pre-eclampsia? Biochem. Soc. Trans. Dec., 37 (Pt 6): 12, 2009. 9- LIM J., KIM Sh and PARK S.: Effective prediction of preeclampsia by a combined ratio of angiogenesis-related factors. N. Engl. J. Med., 355 (17): 1840, 2006. 10- LEVINE R., LAM Ch and QUIN C.: sEng and other circulating anti-angiogenic factors in preeclampsia. N. Eng. J. Med., 355 (17): 1840, 2006. 11- DE VIVO A., BAVIERA G. and GIORDANO D.: Endoglin, PIGF and sFlt-1 as markers for predicting preeclampsia. Acta. Obst. Gyn., 87: 837-42, 2008. 12- LYNCH A., MURPHY J. and GIBBS R.: The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia. BJOG, 456-62, 2010. 13- MASUYAMA H., SEGAWA T. and SUMIDA Y.: Different profiles of circulating angiogenic factors and adipocytokines between early and late-onset pre-eclampsia. BJOG, 117: 314-20, 2009. 14- JEYABALAN A., MCGONIGAL C. and GILMOUR C.: Circulating and placental endoglin concentrations in pregnancies complicated by intrauterine growth restriction and preeclampsia. Placenta, 29 (6): 555-63, 2008. 15- TROISI R., BRAEKKE K. and KITTLELSEN N.: Blood pressure augmentation and maternal circulating concentration of angiogenic factors at delivery in preeclamptic and uncomplicated pregnancies. AJOG, 199 (6): 653, 2008. 16- REDDY A., SURI S. and SARGENT I.: Maternal circulating levels of activin A, inhibin A, s Flt-1 and endoglin at parturition in normal pregnancy and preeclampsia. PloS One, 4 (2): E 4453, 2009. 17- SAVIDDOU M., NOORI M. and ANDERSON J.: Maternal endothelial function and serum concentrations of placental growth factors & soluble endoglin in women with abnormal placentation. Ultrasound Obst. Gyn., 32: 871-6, 2008.

with history of gest. HTN have approximately 50% lower breast cancer risk compared with women who had uncomplicated pregnancy. We illustrated increased sEng level in patients with gest. HTN compared with uncomplicated pregnancies. The relatively anti-angiogenic profile revealed in PE and gest. HTN may be a marker for persistent difference before and after pregnancy in angiogenic response among these women. Actually Reddy's study [16] has revealed a 55% decline in sEng 24 hrs after placental delivery in PE patients suggesting extra-placental source for the increased levels of sEng. in PE and Saviddou and Nouri, [17] concluded that maternal endothelial dysfunction in PE women persist many years after delivery which support the evidence that the antiangiogenic profile in PE patients carry potential protection against cancer for these patients. What further support this hypothesis are the studies revealing a more pro-angiogenic profile in cancer cases than controls as well as positive association with tumor size and metastasis [15] . A central feature in the pathophysiology of PE is failure of physiological transformation of the spiral arteries which is thought to be responsible for the increased impedence to blood flow in the utero-placental circulation. The resulting poor placentation leads to the release of anti-angiogenic factors in the maternal circulation that cause endothelial cell dysfunction and multiple organ damage. Our study revealed that patients with abnormal Doppler velocimetry had the highest median plasma sEng concentration among all patients. This agrees with Saviddou and Nouri [17] and supports the evidence that PE develops due to placental hypoperfusion. Finally we conclude that altered anti-angiogenic factor might be involved in the pathogenesis of the different hypertensive diseases of pregnancy. sEng had a good discriminatory ability between cases of preeclampsia, gestational hypertension and controls. Our data suggest that sEng level correlates positively with vascular dysfunction as evidenced by increased resistance to umbilical blood flow assessed by Doppler study with high sEng levels. Our results are consistent with the hypothesis that angiogenic balance plays a role in maternal breast cancer risk reduction associated with blood pressure increases in pregnancy. References
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