http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–5 ! 2015 Informa UK Ltd. DOI: 10.3109/09537104.2015.1048213
ORIGINAL ARTICLE
Impact of preeclampsia on megakaryocytopoesis and platelet homeostasis of preterm infants Jie Yang1, HaiChun Zhang1, JianMin Niu2, XiaoPing Mu3, XiaoLing Zhang1, Ying Liu1, JunPing Wang1, & Yunbin Chen1 Department of Neonatology, 2Department of Obstetrics, and 3Department of Clinical Laboratory, Maternal and Children Hospital of Guang Dong Province, Guang Zhou Medical College, Guang Zhou, Guang Dong Province, China
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
1
Abstract
Keywords
The aim of this article is to investigate the megakaryopoyesis and thrombopoiesis in preterm infants born to mothers with preeclampsia and the potential effects mediated by soluble fmslike tyrosine kinase 1 (sFlt1) and thrombopoietin (TPO). A perspective case–control study was performed on 26 cord blood of preterm newborns born to mothers with preeclampsia (PE group) and 26 of preterms born to mothers without preeclampsia (control group). Circulating megakaryocyte count and megakaryocyte colony forming units (CFU-MK) were quantified by whole blood infiltration method and plasma clot culture system, respectively. Platelet activation markers, CD62P and CD63, were estimated by flow cytometry. Immunosorbent assays (ELISA) were employed to estimate plasma levels of sFlt1 and TPO of the two groups. When compared to the controls, infants born to mothers with PE had significantly lower peripheral platelet count (PE vs. controls: 157.9 [44.6] vs. 239.6 [57.5] 109/l, p50.001), circulating MK count (5.8 [1.0] vs. 7.7 [0.9]/ml, p50.001) and CFU-MK (14.1 [2.1] vs. 20.1 [2.8]/1 105 cell, p50.001); greater expressions of CD62P (15.5 [2.3] vs. 11.4 [1.9]% platelets, p50.001) and CD63 (12.3 [2.4] vs. 9.0 [1.6]% platelets, p50.001); increased plasma Flt level (130.1 [8.0] vs. 97.7 [8.7] pg/ml, p50.001) and TPO level (129.5 [17.8] vs. 98.9 [11.8] pg/ml, p50.001). In PE group, sFlt instead of TPO showed a significantly negative relationship with platelet counts, CFU-MK and circulating MK count, a positive relationship with CD62P, CD63 expressions. In control group, both sFlt and TPO did not show any relationship with these parameters. sFlt played important role in megakaryocytopoesis and platelet homeostasis in preterm infants born to mothers with PE. Its mechanism maybe the effect of impaired megakaryocyte formation and increased platelet activation.
Neonates, platelets, preeclampsia, sFlt, thrombopoiesis
Introduction Preeclampsia (PE) is the most common complication of pregnancy, the incidence of which was reported as 2–6% in the United States and 4–18% in the developing nations [1–4]. PE is the second most common obstetric cause of stillbirths and early neonatal deaths in these countries [5]. The major pathological feature of PE is endothelial dysfunction, which causes platelet activation and diffused ischemic disorder [6]. Vascular endothelial growth factors (VEGFs) belong to the platelet-derived growth factor supergene family, and they play central roles in the regulation of angiogenesis and lymphangiogenesis [7, 8]. VEGF-A, the major factor for angiogenesis, binds to two tyrosine kinase (TK) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), the soluble VEGFR-1 (sFlt-1) functions as an endogenous VEGF inhibitor [7, 8]. sFlt-1 is abnormally overexpressed in the placenta of PE patients, resulting in the major symptoms of the disease due to abnormal trapping of VEGFs [9]. Several researchers reported that an abnormal increase in the level of serum sFlt-1 in pregnant mothers is well Correspondence: Dr Yunbin Chen, Department of Neonatology, Maternal and Children Hospital of Guang Dong Province, Guang Zhou Medical College, Guang Zhou, Guang Dong Province, China. Tel: +86-862039158600. Fax: +86-8620-86505085. E-mail: [email protected]
History Received 23 October 2014 Revised 1 May 2015 Accepted 1 May 2015 Published online 17 June 2015
correlated with the degree of PE [10–12]. The condition is usually associated with thrombocytopenia in newborn infants. There is a significantly negative relationship between neonatal platelet count and cord blood sFlt-1 levels [13]. The mechanism responsible for neonatal thrombocytopenia has been considered as the combination of reduced platelet production, reduced circulating MK progenitor and increased immune-mediated platelet consumption [14–16].
Methods Patients The study was approved by the Research Ethics Committee of Guang Zhou Medical College. All infants were recruited from deliveries in Maternal and Children Hospital of Guang Dong Province from Jan 1st, 2012 to Jan 1st, 2013. The experimental group recruitment criteria included: (1) born to mothers with PE; (2) no congenital abnormalities; (3) no any other maternal complications; and (4) preterm. The control group was selected paired to the experimental group case to case. The control group recruitment criteria included: (1) born to mothers without PE and other maternal complications; (2) no congenital abnormalities and (3) matched sex, gestational age at the closest delivery time with the paired experimental group case.
2
J. Yang et al.
The diagnosis of PE was made according to guideline ‘‘Diagnosis and management of preeclampsia and eclampsia’’ established by the American College of Obstetricians and Gynecologists [17]. Neonatal thrombocytopenia was defined arbitrarily as a platelet count of 5150 000 mm3. Neonatal disease such as neonatal respiratory distress syndrome (RDS), hyperglycemia and asphyxia were defined according to Gomella’s Neonatoloy [18]. Measurements
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Blood sampling 300 ml blood was withdrawn from the cord, and then the blood was placed in ethylene-diamine-tetra-acetic acid (EDTA) tube (Sarstedt, Numbrecht, Germany). After mixing gently, 40 ll whole blood was aliquot for the detection of CD62P and CD63 expressions by flow cytometry. After centrifugation at 400 g, plasma was separated from the blood and preserved at 80 C for cytokine level detection. Cell pellet was resuspended in phosphate-buffered saline (PBS) for circulating MK isolation. The flow-through fluid retained from the circulating MK assay was centrifuged. Mononuclear cells (MNC) were obtained by densitygradient centrifugation using Ficoll-Paque (1.077 density Amersham, Pharmacia, Uppsala, Sweden), and megakaryocyte colony-forming-unit assay was then performed. All samples were processed within 1 h of collection. CD62P and CD63 expressions on platelets For staining with fluorochrome labels antibody, 5 ml of whole blood was incubated with monoclonal antibody at saturation concentration in the dark for 30 min at room temperature. FITCconjugated CD61 was used as the ‘‘platelet-specific’’ monoclonal antibody, while PE-conjugated anti-CD62P and anti-CD63 were used as platelet activation marker in dual-color analyses. After incubation, 1 ml 1% paraformaldehyde in PBS (pH 7.4) was added to each tube, and samples were fixed at 4 ºC and acquired by a FACS calibur flow cytometer (Becton Dickinson, CA, USA) within 24 hours of fixation. After identification of platelets by FITC-positive and light scatter gates, binding of CD62P and CD63 was determined by analyzing 20 000 individual platelets. All antibodies were purchased from Pharmingen, San Diego, CA. Blood Flt and thrombopoietin (TPO) level detection Flt-1 and TPO levels were measured using a commercially available ELISA (R&D Systems Quantikine Human TPO ELISA kit, Minneapolis, MN), with a lower limit of detection of 15 pg/ml. Circulating MK count Circulating MK count was estimated. In short, samples were passed through an assembled syringe filter holder (Millipore, Ireland) containing a nucleopore polycarbonate membrane (Millipore) of 5 lm pore diameter at 37 C. This was followed by two 2 ml washes with saline at 37 C. After removal of the membrane, the reverse side was wiped with a saline-dampened tissue and dried rapidly using a gentle heat source and left at room temperature to dry thoroughly overnight [19].
Platelets, Early Online: 1–5
centrifuged at 286 g for 35 minute at 20 C. The upper layer was aspirated without disturbing the layer of the mononuclear cell at the interphase. The interphase cells were carefully transferred to a new 15-ml conical tube, in which PBS containing 0.6% ACD was added. The MNC was mixed and centrifuged at 286 g for 10 minute at 20 C and was washed twice with PBS. Subsequently, the supernatant was completely removed carefully. MNC at a concentration of 1 105/ml were seeded in 10% bovine plasma (Sigma, Saint Louis, MO), 1% fetal calf serum (IMDM, Gibco, Grand Island, NY), 10% bovine serum albumin (BSA, Sigma), 0.34 mg/ml calcium chloride (Sigma), 104M 2-Mercaptoethanol (Sigma), Iscove’s modified Dulbecco’s medium (Gibco), 50 ng/ml TPO (Genzyme, Boston, MA) and 50 ng IL-3/ml (Genzyme). After gentle but thorough mixing, the mixture was placed in a petri dish and incubated at 37 C, in a 5% CO2 incubator for 7 days. A CFU-MK colony was defined as a cluster of three or more CD61 positive cells. Identification of megakaryocytes The petri dish of CFU-MK and the membrane retaining circulating MK were rinsed with 500 ll and 20 ll PBS with 0.5% BSA, respectively. The monoclonal antibody, anti-CD61, conjugated with fluorescein isothiocyanate (FITC) was diluted with PBS in 1:100 dilution. Diluted antibody at 200 and 20 ll were added onto each dish and membrane, respectively. The samples were incubated for 20 minutes and rinsed with 500 and 20 ll PBS with 0.5% BSA. The colonies were examined under inflorescence microscope. Statistical analysis Between-group comparisons were performed using the paired t test. Correlation statistics were analyzed by Spearman correlation coefficients. A p value 0.05 was considered statistically significant. All values were expressed as mean [SEM].
Results Patient characteristics There were totally 10 001 deliveries in Maternal and Children Hospital of Guang Dong Province from Jan 1st, 2012 to Jan 1st, 2013, among which 1265 were preterm and 247 were mothers with PE. Twenty six cases fulfilled the experimental group criteria and 26 paired control cases were recruited. Among 26 mothers with PE, 10 suffered from thrombocytopenia with platelet count less than 100 000 mm3, 9 suffered from proteinuria with excretion 300 mg or more protein in 24 hours. There were no differences in clinical characteristics between two groups. Infants in two groups received similar conventional treatment according to the unit’s protocol. The patients’ clinical characteristics were shown in Table I. Peripheral platelet count When compared to the controls, infants born from mothers with PE had significantly lower peripheral platelet count (PE vs. controls: 157.9 [44.6] vs. 239.6 [57.5] 109/l, p50.001); data were shown in Table II. Megakaryocyte colony forming units (CFU-MK)
CFU-MK with peripheral MNC Samples were diluted with 2–4 volumes of PBS containing 0.6% acid-citrate-dextrose (ACD). The diluted sample was carefully layered over 2 ml Ficoll-Paque (1.077 density Amersham, Pharmacia, Uppsala, Sweden) in a 15-ml conical tube and
When compared to the controls, infants born to mother with PE had significantly lower CFU-MK (PE vs. controls: 14.1 [2.1] vs. 20.1 [2.8]/1 105 cell, p50.001), data were shown in Table II; representative MK progenitors were shown in Figure 1.
Preeclampsia on thrombocytopenia of preterms
DOI: 10.3109/09537104.2015.1048213
3
Table I. Comparison of clinical characteristics between PE and control groups. PE group (n ¼ 26)
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Gestation (weeks) Birth weight (g) Apgar scores: At 1 minute At 5 minute Sex Female Male Diagnosis Respiratory Distress Syndrome Hypoglycemia Respiratory support Fraction of inspired oxygen (FiO2) Blood gas (PH) Platelet count (109/l)
Control group (n ¼ 26)
p
30.4 (2.06) 1.8 (0.47)
30.6 (2.0) 1.9 (0.50)
0.09 0.07
7.0 (5–9) 8.8 (8–10)
7.3 (4–10) 9.1 (8–10)
0.88 0.64
11 15
11 15
22 23 4 3 11 11 0.24 (0.21–0.3) 0.23 (0.21–0.3)
0.84
7.38 (7.31–7.42) 7.38 (7.29–7.42) 0.98 157.9 [44.6] 239.6 [57.5] 50.001
Figure 1. Representative (magnification 40).
morphology
of
MK
progenitors
Table II. Comparison of thrombopoietic parameters between PE and control groups. PE group 9
Platelet count (10 /L) Circulating MK count (ml) CFU-MK (1 105 cell) CD62P (% platelets) CD63 (% platelets) Plasma Flt level (pg/ml) Plasma TPO level (pg/ml)
157.9 5.8 14.1 15.5 12.3 130.1 129.5
[44.6] [1.0] [2.1] [2.3] [2.4] [8.0] [17.8]
Control group 239.6 7.7 20.1 11.4 9.0 97.7 98.9
[57.5] [0.9] [2.8] [1.9] [1.6] [8.7] [11.8]
p 50.001 50.001 50.001 50.001 50.001 50.001 50.001
Data were presented as mean [SEM].
Circulating megakaryocyte count The quantity of circulating megakaryocyte in infants born to mother with PE was significantly lower than the control group (PE vs. controls: 5.8 [1.0] vs. 7.7 [0.9]/ml, p50.001). Data were shown in Table II; representative circulating megakaryocyte was shown in Figure 2.
Figure 2. Representative morphology of circulating megakaryocyte (magnification 40).
CD62P and CD63 expressions on platelets When compared to the controls, infants born to mother with PE had significantly greater expressions of CD62P (PE vs. controls: 15.5 [2.3] vs. 11.4 [1.9]% platelets, p50.001) and CD63 (PE vs. controls: 12.3 [2.4] vs. 9.0 [1.6]% platelets, p50.001). Data were shown in Table II. Plasma Flt and thrombopoietin level Plasma sFlt and TPO levels in infants born to mother with PE were significantly higher than those of control group (sFlt: PE vs. controls, 130.1 [8.0] vs. 97.7 [8.7] pg/ml, p50.001; TPO: PE vs. controls, 129.5 [17.8] vs. 98.9 [11.8] pg/ml, p50.001). Data were shown in Table II. Correlation analysis on sFlt
Figure 3. Correlations between platelet count and plasma sFlt-1 level in PE group. There was a significantly negative relationship between the neonatal platelet count and plasma sFlt-1 levels (p50.01).
In PE group, there was a significantly negative relationship between plasma sFlt-1 level and platelet count, CFU-MK, circulating MK count, respectively (platelet, r ¼ 0.68, p50.001; CFU-MK, r ¼ 0.43; p ¼ 0.04; circulating MK count, r ¼ 0.73, p50.001) and, moreover, a significantly positive relationship between plasma sFlt-1 level and CD 62P, CD63
expression, respectively (CD 62P: r ¼ 0.75; p50.001; CD 63: r ¼ 0.42; p ¼ 0.04). Data were shown in Figures 3, 4 and 5, respectively. However, none of these relationships was observed in the control group.
4
J. Yang et al.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Figure 4. Correlations between CFU-MK, circulating MK count and plasma sFlt-1 level in PE group. There was a significantly negative relationship between CFU-MK, circulating MK count and plasma sFlt-1 levels, respectively, in PE group (CFU-MK, p50.01; circulating MK count, p50.05).
Figure 5. Correlations between platelet activation markers and plasma sFlt-1 level in PE group. There was a significantly negative relationship between CD 62P, CD 63 expression and plasma sFlt-1 level, respectively, in PE group (CD 62P, p50.01; CD 63, p50.05).
Correlation analysis on TPO There were no relationship between TPO and platelet count, CFU-MK, circulating MK count, CD 62P, CD63 expression, respectively, both in the PE and control groups.
Discussion SFlt, functions as an endogenous VEGF inhibitor, excess sFlt-1 may play a important role in PE severity [10, 20–21]. We found that there was a significantly negative relationship between neonatal platelet count and cord blood sFlt-1 levels in infants born to mothers with PE. Our finding was consistent with previous study by Tsao [13]. An autocrine VEGF loop might exist to optimal Mk maturation through Flt1 to affect prenatal thrompoiesis, evidenced by the fact that VEGF enhance Mk polyploidization, on the contrast, Flt1 inhibit Mk polyploidization [22] However, whether there would be a direct relationship between dysfunctional MK maturation and sFlT-1, deserves us to perform in vitro assay of CFU-MK in the presence of exogenously added sFlT-1 in future study. Furthermore, a significantly negative relationship was observed between neonatal MK progenitor, circulating MK count and blood sFlt-1 levels, which showed that sFlt may inhibit MK production in infants born to mothers with PE. A significantly positive relationship was observed between
Platelets, Early Online: 1–5
neonatal CD62P, CD63 expressions and blood sFlt-1 levels, which demonstrated that sFlt may increase platelet activation in infants born to mothers with PE. Endothelial damage and dysfunction are common pathological features in PE. Once endothelial damage occurs, it can initiate the activation of platelets, neutrophils and coagulation system, and promote further vascular damage. The postulated mechanism responsible for PE may be the normal process of trophoblast invasion of maternal spiral arteries impaired [23]. sFlt-1 is expressed most in the trophoblast cells, which act as layer located between the umbilical capillaries on the fetal side and the maternal blood vessels in the placenta [8, 9]. In addition, VEGF signals stimulate eNOS to increase the production of a vasodilator – nitric oxide (NO) in endothelial cells [24]. When pregnant women suffer from PE, an increase in sFlt-1 level may inhibit NO production, which causes vessel constriction [25, 26]. These hint that sFlt-1 plays role in platelet activation. This theory might explain our finding why platelet activation markers, CD62P and CD63 expressions, were intensified in infants of PE mother when compared to control infants, and CD62P and CD63 expressions were significantly correlated with sFlt level. A recent paper has shown that activated platelet in PE could bind to monocytes to form platelet-monocyte aggregates that then can generate sFLt contributing to the increased level of the antiangiogenic factor in PE. It needs us to have further investigation whether sFlT-1 induces platelet activation directly when incubates together then measures platelet activation [27]. In our study, CD62P and CD63 expressions in the PE group were stronger than those in the control group. Our data indicated that platelets in infants born to mothers with PE were more activated than those of control groups. In contrast, Kuhne et al. reported that there was no significance found in the CD62P and CD63 expressions between infants born to preeclamptic mothers and normal infants of healthy mothers [25]. This was not controversial to our finding, since healthy infants were enrolled as control group in Kuhne’s study instead of weight-, gestational age- and sex-matched infants in our study. CD62P and CD63 have important functions in early cell contact with the vessel wall. TPO is the major regulator of circulating platelet count. However, in our study, we found TPO level significantly increased in infants who were born to mothers with PE. In addition, we found no correlation between the TPO level and the neonatal platelet count, circulating MK count and MK progenitor either in the PE and control groups. This indicates that TPO may not be the main regulator of thrombocyte production in preterm infants. TPO of newborn is mainly produced by fetal liver. A previous paper has shown altered intrahepatic hematopoiesis in neonates from women with PE [28]. Our finding was concordant with the previous reports [29]. One possible mechanism is that plasma TPO concentration is dependent on circulating platelet mass [30]. Elevated platelet levels would lead to increased binding of the cytokine to platelet receptors and consequently increased catabolism, resulting in a lower plasma concentration. In contrast, lower platelet levels would lead to decreased uptake and catabolism, hence, a higher plasma TPO concentration. This theory supports our finding. The TPO level in this study was similar with that previously reported on infants born by PE mothers [31]. Despite there was no correlation between platelet count and TPO level either in the PE or control groups, we indeed found increased TPO level in cord blood from the PE group, which may suggest a role of TPO in PE-related thrombocytopenia. Our data showed that circulating MK progenitor and circulating MK cell are reduced in the PE group, reflecting hypogeneration of haematopoiesis occurred in bone marrow. The explanation for our finding is based on the pathological features
Preeclampsia on thrombocytopenia of preterms
DOI: 10.3109/09537104.2015.1048213
of PE. PE is associated with a factor that enhances superoxide production [10], although this factor is still unclear. Once this factor crosses placenta, it may inhibit fetal haematopoiesis. This underlying mechanism is evidenced by the documentation of Murray, on which he showed that thrombocytopenia together with neutropenia was presented in newborn of mother with PE [12, 24]. SFlt played important role in megakaryocytopoesis and platelet homeostasis in preterm infants born to mothers with PE. Its mechanism maybe most likely from the impaired megakaryocyte formation and increased platelet activation. In addition, we used small volume of blood to achieve the whole picture of neonatal thrombopoiesis, our results may provide further insights into the mechanism of thrombocytopenia induced by PE.
13.
14. 15. 16.
17.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Declaration of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing our work.
18. 19.
20.
References 1. Sibai BM. Diagnosis and management of gestational hypertension and preeclampsia. Obstet Gynecol 2003;102:181–192. 2. Thornton C, Dahlen H, Korda A, Hennessy A. The incidence of preeclampsia and eclampsia and associated maternal mortality in Australia from population-linked datasets: 2000–2008. Am J Obstet Gynecol 2013;208:476.e1–5. 3. Vatten LJ, Skjaerven R. Is pre-eclampsia more than one disease? BJOG 2004;111:298–302. 4. Villar J, Betran AP, Gulmezoglu M. Epidemiological basis for the planning of maternal health services. WHO/RHR 2001. 5. Ngoc NT, Merialdi M, Abdel-Aleem H. Causes of stillbirths and early neonatal deaths: Data from 7993 pregnancies in six developing countries. Bull World Health Organ 2006;84:699–705. 6. Stevens DU, Al-Nasiry S, Fajta MM, Bulten J, van Dijk AP, van der Vlugt MJ, Oyen WJ, van Vugt JM, Spaanderman ME. Cardiovascular and thrombogenic risk of decidual vasculopathy in preeclampsia. Am J Obstet Gynecol 2014;210:545–546. 7. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005;438:967–974. 8. Shibuya M. Vascular endothelial growth factor and its receptor system: Physiological functions in angiogenesis and pathological roles in various diseases. J Biochem 2013;153:13–19. 9. McMahon K, Karumanchi SA, Stillman IE, Cummings P, Patton D, Easterling T. Does soluble fms-like tyrosine kinase-1 regulate placental invasion? Insight from the invasive placenta. Am J Obstet Gynecol 2014;210:68.e1–4. 10. Maynard, SEMin JY, Merchan J, Lim K-H, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649–658. 11. Koga K, Osuga Y, Yoshino O, Hirota Y, Ruimeng X, Hirata T, Takeda S, Yano T, Tsutsumi O, Taketani Y. Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia. J Clin Endocrinol Metab 2003;88: 2348–2351. 12. Levine RJ, Maynard SE, Qian C, Lim K-H, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, et al.
21. 22. 23. 24.
25. 26. 27.
28.
29.
30.
31.
5
Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672–683. Tsao P-N, Wei S-C, Su Y-N, Chou HC, Chen CY, Hsieh WS. Excess soluble fms-like tyrosine kinase 1 and low platelet counts in premature neonates of preeclamptic mothers. Pediatrics 2005;116: 468–472. Murray NA, Roberts IA. Circulating megakaryocytes and their progenitors in early thrombocytopenia in preterm neonates. Pediatr Res 1996;40:112–119. Burrows RF, Hunter DJ, Andrew M, Kelton JG. A prospective study investigating the mechanism of thrombocytopenia in preeclampsia. Obstet Gynecol 1987;70:334–338. Samuels P, Main EK, Tomaski A, Mennuti MT, Gabbe SG, Cines DB. Abnormalities in platelet antiglobulin tests in preeclamptic mothers and their neonates. Am J Obstet Gynecol 1987;157: 109–113. ACOG Committee on Obstetric Practice. ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January 2002. Int J Obstet Gynecol 2002;77:67–75. Gomella T, Cummingham M, Fabien E. Neonatology. 6th ed. New York: Lange; 2009. Yang J, Yang M, Xu F, Li K, Lee SK, Ng PC, Tam JS, Yuen PM, Fok TF. Effects of oxygen-induced lung damage on megakaryocytopoiesis and platelet homeostasis in a rat model. Pediatr Res 2003; 54:344–352. Ahmad S, Ahmed A. Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia. Circ Res 2004;95:884–891. Thadhani R, Mutter WP, Wolf M. First trimester placental growth factor and soluble fms-like tyrosine kinase 1 and risk for preeclampsia. J Clin Endocrinol Metab 2004;89:770–775. Casella I, Feccia T, Chelucci C. Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor. Blood 2003;101:1316–1323. Pijnenborg R, Anthony J, Davey DA. Placental bed spiral arteries in the hypertensive disorders of pregnancy. Br J Obstet Gynaecol 1991; 98:648–655. Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: A critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 2002;20: 4368–4380. Kuhne T, Ryan G, Blanchette V. Platelet-surface glycoproteins in healthy and preeclamptic mothers and their newborn infants. Pediatr Res 1996;40:876–880. Abrams C, Shattil SJ. Immunological detection of activated platelets in clinical disorders. Thromb Haemost 1991;65:467–473. Major HD, Campbell RA, Silver RM, Branch DW, Weyrich AS. Synthesis of sFlt-1 by platelet-monocyte aggregates contributes to the pathogenesis of preeclampsia. Am J Obstet Gynecol 2014;210: 547.e1–7. Tamiolakis D, Venizelos I, Lambropoulou M, Efthymiadou A, Arvanitidou V, Tsikouras P, Koutsougeras G, Chimonis G, Karamanidis D, Barbagadaki S. Altered intrahepatic hematopoiesis in neonates from women with pregnancy induced hypertension/preeclampsia. Acta Medica (Hradec Kralove) 2004;47:119–123. Murray NA, Watts TL, Roberts IA. Endogenous thrombopoietin levels and effect of recombinant human thrombopoietin on megakaryocyte precursors in term and preterm babies. Pediatr Res 1998;43:148–151. Kuter DJ, Rosenberg RD. The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood 1995;85: 2720–2730. Tsao PN, Teng RJ, Chou HC, Tsou KI. The thrombopoietin level in the cord blood in premature infants born to mothers with pregnancyinduced hypertension. Biol Neonate 2002;82:217–221.
ORIGINAL ARTICLE
Impact of preeclampsia on megakaryocytopoesis and platelet homeostasis of preterm infants Jie Yang1, HaiChun Zhang1, JianMin Niu2, XiaoPing Mu3, XiaoLing Zhang1, Ying Liu1, JunPing Wang1, & Yunbin Chen1 Department of Neonatology, 2Department of Obstetrics, and 3Department of Clinical Laboratory, Maternal and Children Hospital of Guang Dong Province, Guang Zhou Medical College, Guang Zhou, Guang Dong Province, China
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
1
Abstract
Keywords
The aim of this article is to investigate the megakaryopoyesis and thrombopoiesis in preterm infants born to mothers with preeclampsia and the potential effects mediated by soluble fmslike tyrosine kinase 1 (sFlt1) and thrombopoietin (TPO). A perspective case–control study was performed on 26 cord blood of preterm newborns born to mothers with preeclampsia (PE group) and 26 of preterms born to mothers without preeclampsia (control group). Circulating megakaryocyte count and megakaryocyte colony forming units (CFU-MK) were quantified by whole blood infiltration method and plasma clot culture system, respectively. Platelet activation markers, CD62P and CD63, were estimated by flow cytometry. Immunosorbent assays (ELISA) were employed to estimate plasma levels of sFlt1 and TPO of the two groups. When compared to the controls, infants born to mothers with PE had significantly lower peripheral platelet count (PE vs. controls: 157.9 [44.6] vs. 239.6 [57.5] 109/l, p50.001), circulating MK count (5.8 [1.0] vs. 7.7 [0.9]/ml, p50.001) and CFU-MK (14.1 [2.1] vs. 20.1 [2.8]/1 105 cell, p50.001); greater expressions of CD62P (15.5 [2.3] vs. 11.4 [1.9]% platelets, p50.001) and CD63 (12.3 [2.4] vs. 9.0 [1.6]% platelets, p50.001); increased plasma Flt level (130.1 [8.0] vs. 97.7 [8.7] pg/ml, p50.001) and TPO level (129.5 [17.8] vs. 98.9 [11.8] pg/ml, p50.001). In PE group, sFlt instead of TPO showed a significantly negative relationship with platelet counts, CFU-MK and circulating MK count, a positive relationship with CD62P, CD63 expressions. In control group, both sFlt and TPO did not show any relationship with these parameters. sFlt played important role in megakaryocytopoesis and platelet homeostasis in preterm infants born to mothers with PE. Its mechanism maybe the effect of impaired megakaryocyte formation and increased platelet activation.
Neonates, platelets, preeclampsia, sFlt, thrombopoiesis
Introduction Preeclampsia (PE) is the most common complication of pregnancy, the incidence of which was reported as 2–6% in the United States and 4–18% in the developing nations [1–4]. PE is the second most common obstetric cause of stillbirths and early neonatal deaths in these countries [5]. The major pathological feature of PE is endothelial dysfunction, which causes platelet activation and diffused ischemic disorder [6]. Vascular endothelial growth factors (VEGFs) belong to the platelet-derived growth factor supergene family, and they play central roles in the regulation of angiogenesis and lymphangiogenesis [7, 8]. VEGF-A, the major factor for angiogenesis, binds to two tyrosine kinase (TK) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), the soluble VEGFR-1 (sFlt-1) functions as an endogenous VEGF inhibitor [7, 8]. sFlt-1 is abnormally overexpressed in the placenta of PE patients, resulting in the major symptoms of the disease due to abnormal trapping of VEGFs [9]. Several researchers reported that an abnormal increase in the level of serum sFlt-1 in pregnant mothers is well Correspondence: Dr Yunbin Chen, Department of Neonatology, Maternal and Children Hospital of Guang Dong Province, Guang Zhou Medical College, Guang Zhou, Guang Dong Province, China. Tel: +86-862039158600. Fax: +86-8620-86505085. E-mail: [email protected]
History Received 23 October 2014 Revised 1 May 2015 Accepted 1 May 2015 Published online 17 June 2015
correlated with the degree of PE [10–12]. The condition is usually associated with thrombocytopenia in newborn infants. There is a significantly negative relationship between neonatal platelet count and cord blood sFlt-1 levels [13]. The mechanism responsible for neonatal thrombocytopenia has been considered as the combination of reduced platelet production, reduced circulating MK progenitor and increased immune-mediated platelet consumption [14–16].
Methods Patients The study was approved by the Research Ethics Committee of Guang Zhou Medical College. All infants were recruited from deliveries in Maternal and Children Hospital of Guang Dong Province from Jan 1st, 2012 to Jan 1st, 2013. The experimental group recruitment criteria included: (1) born to mothers with PE; (2) no congenital abnormalities; (3) no any other maternal complications; and (4) preterm. The control group was selected paired to the experimental group case to case. The control group recruitment criteria included: (1) born to mothers without PE and other maternal complications; (2) no congenital abnormalities and (3) matched sex, gestational age at the closest delivery time with the paired experimental group case.
2
J. Yang et al.
The diagnosis of PE was made according to guideline ‘‘Diagnosis and management of preeclampsia and eclampsia’’ established by the American College of Obstetricians and Gynecologists [17]. Neonatal thrombocytopenia was defined arbitrarily as a platelet count of 5150 000 mm3. Neonatal disease such as neonatal respiratory distress syndrome (RDS), hyperglycemia and asphyxia were defined according to Gomella’s Neonatoloy [18]. Measurements
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Blood sampling 300 ml blood was withdrawn from the cord, and then the blood was placed in ethylene-diamine-tetra-acetic acid (EDTA) tube (Sarstedt, Numbrecht, Germany). After mixing gently, 40 ll whole blood was aliquot for the detection of CD62P and CD63 expressions by flow cytometry. After centrifugation at 400 g, plasma was separated from the blood and preserved at 80 C for cytokine level detection. Cell pellet was resuspended in phosphate-buffered saline (PBS) for circulating MK isolation. The flow-through fluid retained from the circulating MK assay was centrifuged. Mononuclear cells (MNC) were obtained by densitygradient centrifugation using Ficoll-Paque (1.077 density Amersham, Pharmacia, Uppsala, Sweden), and megakaryocyte colony-forming-unit assay was then performed. All samples were processed within 1 h of collection. CD62P and CD63 expressions on platelets For staining with fluorochrome labels antibody, 5 ml of whole blood was incubated with monoclonal antibody at saturation concentration in the dark for 30 min at room temperature. FITCconjugated CD61 was used as the ‘‘platelet-specific’’ monoclonal antibody, while PE-conjugated anti-CD62P and anti-CD63 were used as platelet activation marker in dual-color analyses. After incubation, 1 ml 1% paraformaldehyde in PBS (pH 7.4) was added to each tube, and samples were fixed at 4 ºC and acquired by a FACS calibur flow cytometer (Becton Dickinson, CA, USA) within 24 hours of fixation. After identification of platelets by FITC-positive and light scatter gates, binding of CD62P and CD63 was determined by analyzing 20 000 individual platelets. All antibodies were purchased from Pharmingen, San Diego, CA. Blood Flt and thrombopoietin (TPO) level detection Flt-1 and TPO levels were measured using a commercially available ELISA (R&D Systems Quantikine Human TPO ELISA kit, Minneapolis, MN), with a lower limit of detection of 15 pg/ml. Circulating MK count Circulating MK count was estimated. In short, samples were passed through an assembled syringe filter holder (Millipore, Ireland) containing a nucleopore polycarbonate membrane (Millipore) of 5 lm pore diameter at 37 C. This was followed by two 2 ml washes with saline at 37 C. After removal of the membrane, the reverse side was wiped with a saline-dampened tissue and dried rapidly using a gentle heat source and left at room temperature to dry thoroughly overnight [19].
Platelets, Early Online: 1–5
centrifuged at 286 g for 35 minute at 20 C. The upper layer was aspirated without disturbing the layer of the mononuclear cell at the interphase. The interphase cells were carefully transferred to a new 15-ml conical tube, in which PBS containing 0.6% ACD was added. The MNC was mixed and centrifuged at 286 g for 10 minute at 20 C and was washed twice with PBS. Subsequently, the supernatant was completely removed carefully. MNC at a concentration of 1 105/ml were seeded in 10% bovine plasma (Sigma, Saint Louis, MO), 1% fetal calf serum (IMDM, Gibco, Grand Island, NY), 10% bovine serum albumin (BSA, Sigma), 0.34 mg/ml calcium chloride (Sigma), 104M 2-Mercaptoethanol (Sigma), Iscove’s modified Dulbecco’s medium (Gibco), 50 ng/ml TPO (Genzyme, Boston, MA) and 50 ng IL-3/ml (Genzyme). After gentle but thorough mixing, the mixture was placed in a petri dish and incubated at 37 C, in a 5% CO2 incubator for 7 days. A CFU-MK colony was defined as a cluster of three or more CD61 positive cells. Identification of megakaryocytes The petri dish of CFU-MK and the membrane retaining circulating MK were rinsed with 500 ll and 20 ll PBS with 0.5% BSA, respectively. The monoclonal antibody, anti-CD61, conjugated with fluorescein isothiocyanate (FITC) was diluted with PBS in 1:100 dilution. Diluted antibody at 200 and 20 ll were added onto each dish and membrane, respectively. The samples were incubated for 20 minutes and rinsed with 500 and 20 ll PBS with 0.5% BSA. The colonies were examined under inflorescence microscope. Statistical analysis Between-group comparisons were performed using the paired t test. Correlation statistics were analyzed by Spearman correlation coefficients. A p value 0.05 was considered statistically significant. All values were expressed as mean [SEM].
Results Patient characteristics There were totally 10 001 deliveries in Maternal and Children Hospital of Guang Dong Province from Jan 1st, 2012 to Jan 1st, 2013, among which 1265 were preterm and 247 were mothers with PE. Twenty six cases fulfilled the experimental group criteria and 26 paired control cases were recruited. Among 26 mothers with PE, 10 suffered from thrombocytopenia with platelet count less than 100 000 mm3, 9 suffered from proteinuria with excretion 300 mg or more protein in 24 hours. There were no differences in clinical characteristics between two groups. Infants in two groups received similar conventional treatment according to the unit’s protocol. The patients’ clinical characteristics were shown in Table I. Peripheral platelet count When compared to the controls, infants born from mothers with PE had significantly lower peripheral platelet count (PE vs. controls: 157.9 [44.6] vs. 239.6 [57.5] 109/l, p50.001); data were shown in Table II. Megakaryocyte colony forming units (CFU-MK)
CFU-MK with peripheral MNC Samples were diluted with 2–4 volumes of PBS containing 0.6% acid-citrate-dextrose (ACD). The diluted sample was carefully layered over 2 ml Ficoll-Paque (1.077 density Amersham, Pharmacia, Uppsala, Sweden) in a 15-ml conical tube and
When compared to the controls, infants born to mother with PE had significantly lower CFU-MK (PE vs. controls: 14.1 [2.1] vs. 20.1 [2.8]/1 105 cell, p50.001), data were shown in Table II; representative MK progenitors were shown in Figure 1.
Preeclampsia on thrombocytopenia of preterms
DOI: 10.3109/09537104.2015.1048213
3
Table I. Comparison of clinical characteristics between PE and control groups. PE group (n ¼ 26)
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Gestation (weeks) Birth weight (g) Apgar scores: At 1 minute At 5 minute Sex Female Male Diagnosis Respiratory Distress Syndrome Hypoglycemia Respiratory support Fraction of inspired oxygen (FiO2) Blood gas (PH) Platelet count (109/l)
Control group (n ¼ 26)
p
30.4 (2.06) 1.8 (0.47)
30.6 (2.0) 1.9 (0.50)
0.09 0.07
7.0 (5–9) 8.8 (8–10)
7.3 (4–10) 9.1 (8–10)
0.88 0.64
11 15
11 15
22 23 4 3 11 11 0.24 (0.21–0.3) 0.23 (0.21–0.3)
0.84
7.38 (7.31–7.42) 7.38 (7.29–7.42) 0.98 157.9 [44.6] 239.6 [57.5] 50.001
Figure 1. Representative (magnification 40).
morphology
of
MK
progenitors
Table II. Comparison of thrombopoietic parameters between PE and control groups. PE group 9
Platelet count (10 /L) Circulating MK count (ml) CFU-MK (1 105 cell) CD62P (% platelets) CD63 (% platelets) Plasma Flt level (pg/ml) Plasma TPO level (pg/ml)
157.9 5.8 14.1 15.5 12.3 130.1 129.5
[44.6] [1.0] [2.1] [2.3] [2.4] [8.0] [17.8]
Control group 239.6 7.7 20.1 11.4 9.0 97.7 98.9
[57.5] [0.9] [2.8] [1.9] [1.6] [8.7] [11.8]
p 50.001 50.001 50.001 50.001 50.001 50.001 50.001
Data were presented as mean [SEM].
Circulating megakaryocyte count The quantity of circulating megakaryocyte in infants born to mother with PE was significantly lower than the control group (PE vs. controls: 5.8 [1.0] vs. 7.7 [0.9]/ml, p50.001). Data were shown in Table II; representative circulating megakaryocyte was shown in Figure 2.
Figure 2. Representative morphology of circulating megakaryocyte (magnification 40).
CD62P and CD63 expressions on platelets When compared to the controls, infants born to mother with PE had significantly greater expressions of CD62P (PE vs. controls: 15.5 [2.3] vs. 11.4 [1.9]% platelets, p50.001) and CD63 (PE vs. controls: 12.3 [2.4] vs. 9.0 [1.6]% platelets, p50.001). Data were shown in Table II. Plasma Flt and thrombopoietin level Plasma sFlt and TPO levels in infants born to mother with PE were significantly higher than those of control group (sFlt: PE vs. controls, 130.1 [8.0] vs. 97.7 [8.7] pg/ml, p50.001; TPO: PE vs. controls, 129.5 [17.8] vs. 98.9 [11.8] pg/ml, p50.001). Data were shown in Table II. Correlation analysis on sFlt
Figure 3. Correlations between platelet count and plasma sFlt-1 level in PE group. There was a significantly negative relationship between the neonatal platelet count and plasma sFlt-1 levels (p50.01).
In PE group, there was a significantly negative relationship between plasma sFlt-1 level and platelet count, CFU-MK, circulating MK count, respectively (platelet, r ¼ 0.68, p50.001; CFU-MK, r ¼ 0.43; p ¼ 0.04; circulating MK count, r ¼ 0.73, p50.001) and, moreover, a significantly positive relationship between plasma sFlt-1 level and CD 62P, CD63
expression, respectively (CD 62P: r ¼ 0.75; p50.001; CD 63: r ¼ 0.42; p ¼ 0.04). Data were shown in Figures 3, 4 and 5, respectively. However, none of these relationships was observed in the control group.
4
J. Yang et al.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Figure 4. Correlations between CFU-MK, circulating MK count and plasma sFlt-1 level in PE group. There was a significantly negative relationship between CFU-MK, circulating MK count and plasma sFlt-1 levels, respectively, in PE group (CFU-MK, p50.01; circulating MK count, p50.05).
Figure 5. Correlations between platelet activation markers and plasma sFlt-1 level in PE group. There was a significantly negative relationship between CD 62P, CD 63 expression and plasma sFlt-1 level, respectively, in PE group (CD 62P, p50.01; CD 63, p50.05).
Correlation analysis on TPO There were no relationship between TPO and platelet count, CFU-MK, circulating MK count, CD 62P, CD63 expression, respectively, both in the PE and control groups.
Discussion SFlt, functions as an endogenous VEGF inhibitor, excess sFlt-1 may play a important role in PE severity [10, 20–21]. We found that there was a significantly negative relationship between neonatal platelet count and cord blood sFlt-1 levels in infants born to mothers with PE. Our finding was consistent with previous study by Tsao [13]. An autocrine VEGF loop might exist to optimal Mk maturation through Flt1 to affect prenatal thrompoiesis, evidenced by the fact that VEGF enhance Mk polyploidization, on the contrast, Flt1 inhibit Mk polyploidization [22] However, whether there would be a direct relationship between dysfunctional MK maturation and sFlT-1, deserves us to perform in vitro assay of CFU-MK in the presence of exogenously added sFlT-1 in future study. Furthermore, a significantly negative relationship was observed between neonatal MK progenitor, circulating MK count and blood sFlt-1 levels, which showed that sFlt may inhibit MK production in infants born to mothers with PE. A significantly positive relationship was observed between
Platelets, Early Online: 1–5
neonatal CD62P, CD63 expressions and blood sFlt-1 levels, which demonstrated that sFlt may increase platelet activation in infants born to mothers with PE. Endothelial damage and dysfunction are common pathological features in PE. Once endothelial damage occurs, it can initiate the activation of platelets, neutrophils and coagulation system, and promote further vascular damage. The postulated mechanism responsible for PE may be the normal process of trophoblast invasion of maternal spiral arteries impaired [23]. sFlt-1 is expressed most in the trophoblast cells, which act as layer located between the umbilical capillaries on the fetal side and the maternal blood vessels in the placenta [8, 9]. In addition, VEGF signals stimulate eNOS to increase the production of a vasodilator – nitric oxide (NO) in endothelial cells [24]. When pregnant women suffer from PE, an increase in sFlt-1 level may inhibit NO production, which causes vessel constriction [25, 26]. These hint that sFlt-1 plays role in platelet activation. This theory might explain our finding why platelet activation markers, CD62P and CD63 expressions, were intensified in infants of PE mother when compared to control infants, and CD62P and CD63 expressions were significantly correlated with sFlt level. A recent paper has shown that activated platelet in PE could bind to monocytes to form platelet-monocyte aggregates that then can generate sFLt contributing to the increased level of the antiangiogenic factor in PE. It needs us to have further investigation whether sFlT-1 induces platelet activation directly when incubates together then measures platelet activation [27]. In our study, CD62P and CD63 expressions in the PE group were stronger than those in the control group. Our data indicated that platelets in infants born to mothers with PE were more activated than those of control groups. In contrast, Kuhne et al. reported that there was no significance found in the CD62P and CD63 expressions between infants born to preeclamptic mothers and normal infants of healthy mothers [25]. This was not controversial to our finding, since healthy infants were enrolled as control group in Kuhne’s study instead of weight-, gestational age- and sex-matched infants in our study. CD62P and CD63 have important functions in early cell contact with the vessel wall. TPO is the major regulator of circulating platelet count. However, in our study, we found TPO level significantly increased in infants who were born to mothers with PE. In addition, we found no correlation between the TPO level and the neonatal platelet count, circulating MK count and MK progenitor either in the PE and control groups. This indicates that TPO may not be the main regulator of thrombocyte production in preterm infants. TPO of newborn is mainly produced by fetal liver. A previous paper has shown altered intrahepatic hematopoiesis in neonates from women with PE [28]. Our finding was concordant with the previous reports [29]. One possible mechanism is that plasma TPO concentration is dependent on circulating platelet mass [30]. Elevated platelet levels would lead to increased binding of the cytokine to platelet receptors and consequently increased catabolism, resulting in a lower plasma concentration. In contrast, lower platelet levels would lead to decreased uptake and catabolism, hence, a higher plasma TPO concentration. This theory supports our finding. The TPO level in this study was similar with that previously reported on infants born by PE mothers [31]. Despite there was no correlation between platelet count and TPO level either in the PE or control groups, we indeed found increased TPO level in cord blood from the PE group, which may suggest a role of TPO in PE-related thrombocytopenia. Our data showed that circulating MK progenitor and circulating MK cell are reduced in the PE group, reflecting hypogeneration of haematopoiesis occurred in bone marrow. The explanation for our finding is based on the pathological features
Preeclampsia on thrombocytopenia of preterms
DOI: 10.3109/09537104.2015.1048213
of PE. PE is associated with a factor that enhances superoxide production [10], although this factor is still unclear. Once this factor crosses placenta, it may inhibit fetal haematopoiesis. This underlying mechanism is evidenced by the documentation of Murray, on which he showed that thrombocytopenia together with neutropenia was presented in newborn of mother with PE [12, 24]. SFlt played important role in megakaryocytopoesis and platelet homeostasis in preterm infants born to mothers with PE. Its mechanism maybe most likely from the impaired megakaryocyte formation and increased platelet activation. In addition, we used small volume of blood to achieve the whole picture of neonatal thrombopoiesis, our results may provide further insights into the mechanism of thrombocytopenia induced by PE.
13.
14. 15. 16.
17.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Declaration of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing our work.
18. 19.
20.
References 1. Sibai BM. Diagnosis and management of gestational hypertension and preeclampsia. Obstet Gynecol 2003;102:181–192. 2. Thornton C, Dahlen H, Korda A, Hennessy A. The incidence of preeclampsia and eclampsia and associated maternal mortality in Australia from population-linked datasets: 2000–2008. Am J Obstet Gynecol 2013;208:476.e1–5. 3. Vatten LJ, Skjaerven R. Is pre-eclampsia more than one disease? BJOG 2004;111:298–302. 4. Villar J, Betran AP, Gulmezoglu M. Epidemiological basis for the planning of maternal health services. WHO/RHR 2001. 5. Ngoc NT, Merialdi M, Abdel-Aleem H. Causes of stillbirths and early neonatal deaths: Data from 7993 pregnancies in six developing countries. Bull World Health Organ 2006;84:699–705. 6. Stevens DU, Al-Nasiry S, Fajta MM, Bulten J, van Dijk AP, van der Vlugt MJ, Oyen WJ, van Vugt JM, Spaanderman ME. Cardiovascular and thrombogenic risk of decidual vasculopathy in preeclampsia. Am J Obstet Gynecol 2014;210:545–546. 7. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005;438:967–974. 8. Shibuya M. Vascular endothelial growth factor and its receptor system: Physiological functions in angiogenesis and pathological roles in various diseases. J Biochem 2013;153:13–19. 9. McMahon K, Karumanchi SA, Stillman IE, Cummings P, Patton D, Easterling T. Does soluble fms-like tyrosine kinase-1 regulate placental invasion? Insight from the invasive placenta. Am J Obstet Gynecol 2014;210:68.e1–4. 10. Maynard, SEMin JY, Merchan J, Lim K-H, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649–658. 11. Koga K, Osuga Y, Yoshino O, Hirota Y, Ruimeng X, Hirata T, Takeda S, Yano T, Tsutsumi O, Taketani Y. Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia. J Clin Endocrinol Metab 2003;88: 2348–2351. 12. Levine RJ, Maynard SE, Qian C, Lim K-H, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, et al.
21. 22. 23. 24.
25. 26. 27.
28.
29.
30.
31.
5
Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672–683. Tsao P-N, Wei S-C, Su Y-N, Chou HC, Chen CY, Hsieh WS. Excess soluble fms-like tyrosine kinase 1 and low platelet counts in premature neonates of preeclamptic mothers. Pediatrics 2005;116: 468–472. Murray NA, Roberts IA. Circulating megakaryocytes and their progenitors in early thrombocytopenia in preterm neonates. Pediatr Res 1996;40:112–119. Burrows RF, Hunter DJ, Andrew M, Kelton JG. A prospective study investigating the mechanism of thrombocytopenia in preeclampsia. Obstet Gynecol 1987;70:334–338. Samuels P, Main EK, Tomaski A, Mennuti MT, Gabbe SG, Cines DB. Abnormalities in platelet antiglobulin tests in preeclamptic mothers and their neonates. Am J Obstet Gynecol 1987;157: 109–113. ACOG Committee on Obstetric Practice. ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January 2002. Int J Obstet Gynecol 2002;77:67–75. Gomella T, Cummingham M, Fabien E. Neonatology. 6th ed. New York: Lange; 2009. Yang J, Yang M, Xu F, Li K, Lee SK, Ng PC, Tam JS, Yuen PM, Fok TF. Effects of oxygen-induced lung damage on megakaryocytopoiesis and platelet homeostasis in a rat model. Pediatr Res 2003; 54:344–352. Ahmad S, Ahmed A. Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia. Circ Res 2004;95:884–891. Thadhani R, Mutter WP, Wolf M. First trimester placental growth factor and soluble fms-like tyrosine kinase 1 and risk for preeclampsia. J Clin Endocrinol Metab 2004;89:770–775. Casella I, Feccia T, Chelucci C. Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor. Blood 2003;101:1316–1323. Pijnenborg R, Anthony J, Davey DA. Placental bed spiral arteries in the hypertensive disorders of pregnancy. Br J Obstet Gynaecol 1991; 98:648–655. Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: A critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 2002;20: 4368–4380. Kuhne T, Ryan G, Blanchette V. Platelet-surface glycoproteins in healthy and preeclamptic mothers and their newborn infants. Pediatr Res 1996;40:876–880. Abrams C, Shattil SJ. Immunological detection of activated platelets in clinical disorders. Thromb Haemost 1991;65:467–473. Major HD, Campbell RA, Silver RM, Branch DW, Weyrich AS. Synthesis of sFlt-1 by platelet-monocyte aggregates contributes to the pathogenesis of preeclampsia. Am J Obstet Gynecol 2014;210: 547.e1–7. Tamiolakis D, Venizelos I, Lambropoulou M, Efthymiadou A, Arvanitidou V, Tsikouras P, Koutsougeras G, Chimonis G, Karamanidis D, Barbagadaki S. Altered intrahepatic hematopoiesis in neonates from women with pregnancy induced hypertension/preeclampsia. Acta Medica (Hradec Kralove) 2004;47:119–123. Murray NA, Watts TL, Roberts IA. Endogenous thrombopoietin levels and effect of recombinant human thrombopoietin on megakaryocyte precursors in term and preterm babies. Pediatr Res 1998;43:148–151. Kuter DJ, Rosenberg RD. The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood 1995;85: 2720–2730. Tsao PN, Teng RJ, Chou HC, Tsou KI. The thrombopoietin level in the cord blood in premature infants born to mothers with pregnancyinduced hypertension. Biol Neonate 2002;82:217–221.
