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Proliferative responses in the placenta after endotoxin exposure in preterm fetal sheep
European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 152–157 www.elsevier.com/locate/ejogrb
Proliferative responses in the placenta after endotoxin exposure in preterm fetal sheep§ Yves Garnier a,b,*, Mamed Kadyrov c, Markus Gantert a,b, Anke Einig a,c, Werner Rath a, Berthold Huppertz c,d a
Department of Obstetrics and Gynecology, University Hospital RWTH Aachen, Germany b Department of Obstetrics and Gynecology, University Hospital Cologne, Germany c Institute of Anatomy II, University Hospital RWTH Aachen, Germany d Institute of Cell Biology, Histology and Embryology, Medical University Graz, Austria
Received 2 August 2006; received in revised form 5 August 2007; accepted 22 August 2007
Abstract Objectives: Antenatal infections are associated with an increased risk of perinatal morbidity and mortality. Systemic application of endotoxins to the fetus results in an increase in placental vascular resistance and chronic reduction in umbilical blood flow. We studied morphological alterations of the placenta in response to fetal inflammation in the preterm sheep. Study design: Therefore, 14 fetal sheep were chronically instrumented at a mean gestational age of 107 1 days (term is 147 days). Four days after surgery fetuses received 100 ng lipopolysaccharide (LPS; n = 8) or saline (control; n = 6) intravenously. Fetal heart rate and arterial blood pressure were monitored continuously while blood gases and acid–base balance were measured at time points 0, +1, +3, +6, +12, +24, +48 and +72 h. Three days after LPS application placental cotyledons were analyzed by immunohistochemistry and morphometry. Different primary antibodies like AE 1 and AE 3 against cytokeratins were used. Secondary antibodies were visualized with 3-amino-9-ethylcarbazole (AEC) or using the Vectastain kit (Vector Laboratories, Burlingame, CA). Double staining was carried out first by utilizing Vectastain kit (black), followed by AEC staining (red). Counterstaining was performed with haematoxylin. Results: Fetal tachycardia and hypertension were induced transiently during the first 12 h after LPS application. Fetuses suffered from mild hypoxaemia while acidemia was absent. Morphometry revealed a non-significant shift in the relation of maternal and fetal placental compartments towards the maternal parts in response to LPS treatment. Endotoxin induced an increased proliferation in both compartments of the placenta with a 3.2-fold increase on the maternal and a 1.8-fold increase on the fetal side. Conclusions: Systemic endotoxin exposure of the preterm fetal sheep leads to a change in the gross organization of the placenta and changes in the proliferation patterns in both placental compartments. These rearrangements inside the placenta may disturb its organ function and subsequently lead to fetal morbidity associated with the fetal inflammatory response syndrome and chronic placental dysfunction, respectively. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Fetal inflammatory response syndrome; Chorioamnionitis
1. Introduction
§ The paper contains data that has been presented at, and awarded a prize by, the German Society for Gynaecology and Obstetrics in 2004. * Corresponding author at: University Hospital of Cologne, Department of Obstetrics and Gynecology, Kerpenerstrasse 34, D-50924 Cologne, Germany. Tel.: +49 221 478 4910; fax: +49 221 478 4929. E-mail address: [email protected] (Y. Garnier).
Clinical and epidemiological studies provide evidence that chorioamnionitis gives rise to a fetal inflammatory response. The histology of chorioamnionitis is characterized by polymorph nuclear infiltrations in the placenta and the fetal membranes, with its pathogenesis still unclear. An increasing amount of data supports the existence of asymptomatic bacterial colonialization already during the
0301-2115/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2007.08.016
Y. Garnier et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 152–157
first and second trimester of pregnancy. This silent inflammation may inhibit placental angiogenesis and thus modulate the course of the pregnancy in the long term. The progress of infection leads to a fetal systemic inflammatory response (FIRS), which histologically presents as funisitis (omphalovasculitis). When the chorioamnionitis induces a systemic inflammation in the pregnant woman, the clinical picture of an amnion infection syndrome (AIS) develops, including maternal fever and elevation of infection-related proteins in the maternal compartment. This inflammation contributes to neonatal disorders such as periventricular leukomalacia or bronchopulmonary dysplasia [1–6]. Yoon et al. have shown that antenatal exposure to intra-amniotic inflammation and documented fetal inflammatory response such as funisitis are strong and independent risk factors for the subsequent development of cerebral palsy at the age of 3 years [6]. Histologically, chorioamnionitis is defined by the presence of polymorphonuclear infiltrates in the placenta and its membranes. It affects 20% of term pregnancies and up to 60% of preterm pregnancies and is often an occult finding [7]. Grether and Nelson [4] reported that both clinical and histopathological evidence of placental infection were associated with an increased risk of unexplained cerebral palsy (CP) [OR 9.3, 95% CI 2.7–31 for clinical chorioamnionitis (CA); OR 8.9, 95% CI 1.9–40 for histological CA]. Recently, a meta-analysis confirmed the potential association between CA and CP, in both full term and preterm infants [8]. Lipopolysaccharides (LPS) which are released from gramnegative bacteria are known as potent inflammatory agents that have been previously used to induce a fetal inflammatory response. Data in experimental models demonstrate that application of LPS in premature fetal sheep affects fetal cardiovascular function in a dose-dependent manner [3]. Thus in preterm fetal sheep low dose endotoxin application caused a transient fall in umbilico-placental blood flow, dysregulation of cerebral blood flow and subsequent periventricular brain white matter injury [9–12]. The purpose of the present study was to study whether LPS administration as a paradigm for fetoplacental inflammation results in histopathological changes in the gross organization of the placenta in preterm fetal sheep. These observations may contribute to the understanding how the long-lasting cascades from endotoxin exposure to periventricular leukomalacia or bronchopulmonary dysplasia work out and may give a key function to the placenta.
2. Materials and methods 2.1. Animal preparation The protocols were approved by the Animal Medical Ethics Committee of Maastricht University and met the guidelines of the responsible governmental agencies.
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Fourteen fetal sheep were chronically instrumented at a mean gestational age of 107 1 days (term is 147 days) as described recently [9–11]. Briefly, all ewes underwent surgery with the use of sterile techniques while they were under general anesthesia (1 g thiopental sodium/70 kg intravenously for induction, 0.5–1.0% halothane in a 1:1 nitrous oxide and oxygen mixture for maintenance). The ewes received 1 g ampicilline (Pentrexyl1; Bristol-Myers, Woerden, The Netherlands) s.c. and 10 mg buprenorphin (Temgesic1; Schering, The Netherlands) per kg body weight twice a day for three consecutive postoperative days. A midline abdominal incision was performed. The fetal limbs were identified and exteriorized through an incision in the uterus. Polyvinyl catheters (Maxxim Medical BV, Den Bosch, The Netherlands) with 0.75 mm inner diameter and 1.25 mm outer diameter were inserted via a tibial fetal vein and artery of each hindleg and advanced into the caudal vena cava and abdominal aorta, respectively. The fetal skin was closed with cyanoacrylate glue (Cyanolit, Japan). An intrauterine pressure catheter was placed, and the uterus was sutured after replacing the lost amniotic fluid with a 0.9% saline solution at 39 8C. Catheters were filled with heparin (100 IU/ml; Heparin-Natrium1, Braun, Melsungen, Germany) and exteriorized through a small incision in the flank of the ewe. Catheters were protected by a pouch sewn to the skin of the ewe’s flank. A recovery period of 4 days followed the operation before experiments were started. Ewes were housed in individual cages and had free access to food and water. 2.2. Experimental protocol The experiments were performed 4 days after surgery at 111 1 days. The fetal inflammatory response was induced by endotoxin treatment (100 ng LPS (n = 8) i.v.; derived from E. coli; O127:B8, Sigma–Aldrich) while the control group (n = 6) received 2 ml 0.9% saline i.v. The total number of animals included in the experiment was 14. Blood samples were taken at various time points before (1 h) and after (+1, +3, +6, +12, +24, +48, +72 h) LPS injection from the fetal descending aorta and analyzed for blood gases, acid base balance (AVL 993, Radiometer, Copenhagen, Denmark), hemoglobin concentration and arterial oxygen saturation of hemoglobin (OSM 2 Hemoximeter, Radiometer, Copenhagen, Denmark). Three days after endotoxin administration the animals were anesthetized with sodium pentobarbitone (1 g/70 kg intravenously for induction, 0.5–1.0% halothane in a 1:1 nitrous oxide and oxygen mixture for maintenance). The right atrium and both jugular veins were incised. After heparin infusion (10.000 IU), each animal was perfusionfixed through a needle placed in the left ventricle with 500 ml of buffered paraformaldehyde (4%) until the fluid efflux from the jugular veins was clear. Ewes and fetuses were killed by a maternal overdose of sodium pentobarbitone (150 mg/kg). Placentomes including the maternal
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caruncle and the fetal cotyledon were weighed and fixed for 24 h in phosphate-buffered formaldehyde (4%). Then the specimens (one half of a placentome, from the maternal to the fetal side) were paraffin-embedded. Standard hematoxylin and eosin stains were used to assess histological structures. 2.3. Immunohistochemistry Routine immunohistochemical procedures were performed on serial sections of 5 mm thickness with anticytokeratin type I (used as epithelial marker; dilution 1:100; clone AE1; Serotec). Other antibodies directed against cytokeratins 7, 18, 19, or other pan cytokeratin antibodies such as clone MNF 116 did not reveal any reliable staining. Anti-Ki-67 was used as a proliferation marker (dilution 1:30; clone Mib-1; Dako). Binding of species-specific biotinylated secondary antibodies was visualized with 3amino-9-ethylcarbazole (AEC). For quantification of cell numbers double staining was performed with anti-Ki-67 first utilizing the Vectastain kit (Vector Laboratories, Burlingame, CA) (black) and then anti-cytokeratin type I with AEC (red). Sections were counterstained with haematoxylin. Negative control reactions omitting the primary antibodies or using an IgG1-negative control antibody (DAK-GO1; Dako) displayed no staining.
2.5. Statistical analysis 2.5.1. Physiologic variables and blood gas parameters Statistical analysis was performed by Super Anova Statistical Package (Abacus, Inc., CA, USA). Games– Howell-test was used as post hoc testing procedure. Statistical significance was set at p < 0.05. All data are expressed as mean values S.D. 2.5.2. Histology Placental cellular proliferation and the amount of fetal versus maternal tissues were counted by an investigator (MK) blinded to the experimental protocol. Numerical density and rates of Mib-1 positive cells were calculated for every single section. Then mean and S.D. were calculated for every single section and then for every placenta before calculating mean and S.D. for each group and each investigated variable. Comparisons between groups were carried out using one-way analysis of variance (ANOVA) and post hoc Bonferroni’s t-test. Statistical significance was set at p < 0.05.
3. Results 3.1. Fetal heart rate, blood pressure, blood gases and acid–base balance
2.4. Quantification Measurements were performed on a stereological workstation (Olympus CAST grid; Olympus, Albertslund, Denmark; Olympus BX 50 microscope), and sections were visualized via a CCD color video camera (600 800 pixels; JAI, Glostrup, Denmark) on a 17 in. monitor. On sections stained with anti-cytokeratin type I we were able to differentiate between maternal and fetal tissues. Using a low magnification (lens: 1.25; PlanApo) the area of interest covering the whole placentome was delineated. Using systematic random sampling a computer program selected the counting fields within the area of interest. Fifty-five to 150 fields (depending on the size of the area of interest) were evaluated per section (lens: 20; UPlanApo, NA = 0.70) by taking a grid with 16 crosses and counting the number of crosses per fetal or maternal tissue. On serial sections double-stained with anti-cytokeratin type I and Mib-1, the ‘areas of interest’ were delineated again, covering the whole placentome. Again, the computer chose the counting fields within the area of interest. Then the number of Mib-1 positive maternal and fetal cells (lens: 20; UPlanApo, NA = 0.70) was counted in 55–150 fields per section. Field sizes were determined and the numerical density of Mib-1 positive cells was calculated as cell profiles per area of the maternal and fetal tissues. Sections from one to four paraffin blocks per placentome were analyzed.
All measurements of FHR, MAP, blood gases and acid base balance during the control period were in the normal range for chronically prepared fetal sheep of this gestational age [9–11]. There were no significant differences between the control and LPS group at this time point (Table 1). MAP in the control group was 41 3 mmHg during normoxia, and did not significantly change during the course of the experiment. In response to LPS exposure MAP increased at +1 h (+21%; p < 0.001) and returned to control levels 4–5 h thereafter. FHR increased 3 and 6 h after LPS injection (+10%, p < 0.05; and +16%, p < 0.01), and was significantly higher than FHR in control animals ( p < 0.01) which did not change significantly from baseline. FHR normalized within 24 h after LPS injection. Oxygen saturation of fetal hemoglobin was transiently reduced in response to endotoxaemia (time point +6 h: 41%; p < 0.05) and slowly recovered during the following 24 h although not completely ( p < 0.05 at 72 h). There were no significant differences in arterial blood pH and blood gas parameters between control and LPS treated animals (Table 1). 3.2. Histology Control animals showed no structural abnormalities or signs of inflammation in both the fetal and maternal compartments of the placenta. LPS treatment caused an increase in proliferation in both compartments, which was visualized by double immunohistochemical staining
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Table 1 Fetal heart rate, mean arterial pressure, acid–base balance and blood gases in control (n = 6) and LPS (E) treated (n = 8) preterm fetal sheep Group
0 h
+1 h
+3 h
+6 h
+12 h
+24 h
+48 h
+72 h
Fetal heart rate (beats per min) C 176 6 172 10 E 179 8 169 12
184 12 193 13y
185 12 207 9§,y
184 10 205 12§,y
171 9 181 8
186 9 177 9
185 13 193 14§,y
Mean arterial blood pressure (mmHg) C 41 3 41 3 E 41 3 46 5§,y
43 3 42 4
43 5 39 4
43 4 39 5
40 4 37 4
45 4 39 5
45 4 34 5y
Arterial oxygen saturation C 49.3 5.6 E 52.5 8.7
51.6 3.4 53.2 7.4
52.8 6.8 41.0 7.3
55.5 5.8 31.2 6.8§,y
48.8 6.3 42.2 7.9
54.3 5.5 46.6 4.9
53.6 12.6 50.5 5.6
56.2 11.9 38.8 9.2§,y
Hemoglobin (mg/dl) C 10.1 0.3 E 10.3 0.5
10.5 0.6 10.9 0.8
10.6 0.8 11.8 1.2§
10.3 0.5 12.5 1.1
10.4 0.6 11.4 1.1
10.0 1.1 10.1 0.9
10.3 0.7 10.3 0.6
10.5 0.5 10.4 1.0
PO2 (mmHg) C 22.0 2.6 E 22.7 2.1
21.8 2.7 23.1 2.9
20.9 2.7 21.0 2.6
22.8 2.4 17.3 3.2
21.9 2.0 20.0 2.9
24.2 1.8 21.5 3.0
24.2 3.4 22.7 3.0
22.7 2.9 22.0 2.9
PCO2 (mmHg) C 54.6 3.0 E 54.0 2.9
56.8 5.7 55.7 2.7
56.5 5.4 59.8 4.8
59.3 5.3 62.9 6.0
47.8 6.2 62.0 6.8
53.0 4.2 55.9 5.8
56.2 4.3 52.0 6.7
62.0 5.0 61.1 8.2
pH C E
7.36 0.02 7.33 0.02
7.37 0.04 7.32 0.05
7.35 0.04 7.32 0.04
7.35 0.02 7.33 0.06
7.34 0.01 7.33 0.04
7.36 0.03 7.31 0.06
7.34 0.04 7.32 0.06
7.35 0.03 7.37 0.04
Values are given as means S.D. § p < 0.01 significant between groups. y p < 0.01 significant vs. control within groups.
(Fig. 1). Immunohistochemical staining using antibodies against cytokeratins revealed a clear staining of the fetal trophoblast with a much weaker staining of the remaining maternal uterine epithelial compartment (Fig. 1). This enabled us to differentiate between both the fetal (delineated by trophoblast) and the maternal compartments. After LPS treatment (Fig. 1B) a clear increase in the number of Mib-1 positive nuclei became obvious compared to control tissues (Fig. 1A). Quantification of Mib-1 positive nuclei revealed a significant increase of positive cells in the maternal (control: 0.225 0.095 cells/field; LPS: 0.730 0.259) as well as in the fetal (control: 2.425 0.418 cells/ field; LPS: 4.400 1.224) compartment (Fig. 2A). In the maternal part of the placenta the level of proliferation
increased 3.2-fold, while in the fetal part the increase was only 1.8-fold (Fig. 2A). At the same time the relation between the volumes of maternal and fetal tissues did not change significantly (Fig. 2B). In control tissues the relative distribution of maternal to fetal tissues was 1.55 0.47, and in LPS treated tissues the relative distribution was 1.43 0.27.
4. Discussion In the present study we have shown that intrauterine exposure of the fetus to endotoxaemia, induced by intravenously applied LPS, results in a fetal inflammatory
Fig. 1. Double immunohistochemistry for cytokeratin and Mib-1. (A) In placental tissues from control animals a normal distribution of proliferating cells in the maternal (M) and fetal (F) compartments of the placenta can be seen. (B) In placental tissues from animals treated with 100 ng LPS an increased proliferation level becomes obvious, both in the maternal (M) as well as in the fetal (F) compartment. Magnification 800.
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Fig. 2. Quantification of changes due to LPS treatment. (A) Quantification of the level of proliferation revealed a 3.2-fold increase in the proliferation within the maternal compartment after treatment with LPS. In the fetal compartment this increase was only 1.8-fold. (B) The relative distribution of maternal and fetal tissues does not change significantly after LPS treatment.
response syndrome, i.e. mild and transient depression of fetal cardiovascular function, e.g. fetal tachycardia and hypertension and mild hypoxaemia. These changes occurred transiently and fetal cardiovascular function completely recovered within 72 h after endotoxaemia was induced. Previously, we have shown that under such circumstances an increase in placental vascular resistance and a reduction in umbilical blood flow are present [9,10]. Moreover, this insult led to an inflammatory response in the fetal brain, with cystic lesions in the periventricular white matter, which are often present in immature fetuses with documented chorioamnionitis or funisitis [11,12]. In the present study low dose LPS administration caused a fetal and placental inflammation, which is common under clinical conditions. It is important to note, that the chronic fetal inflammatory response observed in the present study has been induced by a single-shot dose of endotoxin. Although we have not measured endotoxin plasma levels these have been estimated by concerning the fetal blood volume, plasma volume of the fetal blood volume and the administered LPS doses. Thus, the median endotoxin plasma concentrations were within the range observed in patients
with clinical sepsis [13]. In this context it has to be mentioned that humans are much more sensitive to endotoxin than other species, including the sheep. Moreover, the endotoxic effect of LPS widely varies with the bacterial strain from which it has been isolated. This is the first study to show that fetal endotoxaemia caused an increased proliferative response in both compartments of the sheep placenta. Using morphometry and double immunohistochemistry both the relation between fetal and maternal tissues and the proliferation index within both compartments has been quantified. Using immunohistochemistry stainings of the placenta the present study demonstrates that the three epithelial parts of the sheep placenta, i.e. uninucleate trophoblast cells, binucleate cells and all derivatives of the uterine epithelium can be detected separately. Morphologic changes in the placenta in response to LPS treatment might be induced by different mechanisms. In studies using human placental explants LPS resulted in an increase in the release of prostaglandins (PGF2alpha) and proinflammatory cytokines such as tumor necrosis factoralpha, interleukin (IL)-1beta and IL-10 [14]. These responses were similar to that seen after severe oxidative stress in this model [15]. Oxidative stress, which generates reactive oxygen species (ROS), has been postulated to be involved in the pathophysiology of some complications such as infectious disease and preeclampsia during pregnancy, occasionally leading to placental dysfunction or abruptio placentae. Under these circumstances proliferation and apoptosis are increased in the placenta [15–17]. However, differences of the anatomic settings between the sheep and the human placenta have to be taken into account. While there is a haemochorial placental type in the human, the sheep placenta is of the epitheliochorial type. Changes in the fetal compartment of the placenta should arise irrespective of the type of placentation. As to the changes in the maternal compartment, in both settings any factor has to cross the trophoblast layer to reach the maternal compartment. If there is a connective tissue behind this layer as in the sheep it may result in a local effect. If there is blood as in the human, this may lead to a more systemic response in the human. Duncan et al. have demonstrated that repeated exposure to endotoxin in immature fetal sheep reduced both the placental weight and the average cross-sectional area of placentomes [18]. In this context it is important to note that endotoxin exposure of the immature fetal sheep causes a substantial and long-lasting decrease in umbilico-placental blood flow resulting in mild to sustained fetal hypoxaemia, depending on the protocol used [3,9,10]. It has been reported that placental blood flow began to fall 1 h after LPS and was lowest (40%) at 4–5 h after LPS, while placental vascular resistance rose by 75% during this period [9]. Thereafter, placental blood flow slowly returned to control values at 12– 16 h after endotoxaemia was induced. The increase in umbilico-placental vascular resistance during endotoxaemia might be mediated by endothelin (ET-1). ET-1 is a potent
Y. Garnier et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 152–157
endothelium-derived vasoconstrictive peptide that is able to constrict fetoplacental microcirculation and decrease fetal oxygen consumption in fetal sheep [9,19]. It has been shown previously, that ET-1 is released in response to several stimuli, including hypoxia and endotoxaemia [20]. Among the pathophysiologic conditions known to involve the endothelin system, sepsis and endotoxaemia are producing the highest plasma levels of ET-1 [21]. In the present study we did not measure endothelin serum levels, due to the lack of specificity of the test sera and the instability of the molecule in the laboratory setting. However, bacterial endotoxin and septic conditions are known to increase ET-1 concentrations in fetal umbilical arterial plasma more than fivefold in both pigs and humans, reaching levels close to threshold vasoconstriction [22], while the placental microcirculation is remarkably inert to many vasoconstrictors, including norepinephrine and angiotensin II. Thus we speculate, that ET-1 has a critical role in the control of placental microcirculation and might be the mediator of transitory placental hypoperfusion during endotoxaemia seen in the present study. Moreover, other proinflammatory agents such as IL-1, TNF-alpha and TGF-beta, which are released during endotoxaemia, also increase the production of ET-1 from endothelial cells. This placental inflammatory response might have an impact in the pathogenesis of various pregnancy complications such as preterm labor, preeclampsia, abruptio placentae and intrauterine growth restriction, respectively.
References [1] Adinolfi M. Infectious diseases in pregnancy, cytokines and neurological impairment: an hypothesis. Dev Med Child Neurol 1993;35:549–53. [2] Dammann O, Leviton A. Infection remote from the brain, neonatal white matter damage, and cerebral palsy in the preterm infant. Semin Pediatr Neurol 1998;5:190–201. [3] Garnier Y, Coumans AB, Jensen A, Hasaart TH, Berger R. Infectionrelated perinatal brain injury: the pathogenic role of impaired fetal cardiovascular control. J Soc Gynecol Investig 2003;10:450–9. [4] Grether JK, Nelson KB. Maternal infection and cerebral palsy in infants of normal birth weight. JAMA 1997;278:207–11. [5] Leviton A, Paneth N, Reuss L, et al. Maternal infection, fetal inflammatory response, and brain damage in very low birth weight infants. Pediatr Res 1999;46:566–75. [6] Yoon BH, Romero R, Park JS, et al. Fetal exposure to an intra-amniotic inflammation and the development of cerebral palsy at the age of three years. Am J Obstet Gynecol 2000;182:675–81.
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[7] Lahra MM, Jeffery HE. A fetal response to chorioamnionitis is associated with early survival after preterm birth. Am J Obstet Gynecol 2004;190:147–51. [8] Wu YW, Colford Jr JM. Chorioamnionitis as a risk factor for cerebral palsy: a meta-analysis. JAMA 2000;284:1417–24. [9] Coumans AB, Garnier Y, Supcun S, Jensen A, Berger R, Hasaart TH. The effects of low-dose endotoxin on the umbilicoplacental circulation in preterm sheep. J Soc Gynecol Investig 2004;11:289–93. [10] Garnier Y, Coumans A, Jensen A, Berger R, Hasaart THM. Endotoxaemiamia severely affects circulation during normoxia and asphyxia in immature fetal sheep. J Soc Gynecol Investig 2001;8: 134–42. [11] Garnier Y, Berger R, Alm S, et al. Systemic endotoxin administration results in increased S100B protein blood levels and periventricular brain white matter injury in the preterm fetal sheep. Eur J Obstet Gynecol Reprod Biol 2006;124:15–22. [12] Mallard C, Welin AK, Peebles D, Hagberg H, Kjellmer I. White matter injury following systemic endotoxaemiamia or asphyxia in the fetal sheep. Neurochem Res 2003;28:215–23. [13] Opal SM, Scannon PJ, Vincent JL, White M, Carroll SF, Palardy JE, Parejo NA, Pribble JP, Lemke JH. Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock. J Infect Diss 1999;180:1584–9. [14] Sato TA, Keelan JA, Mitchell MD. Critical paracrine interactions between TNF-alpha and IL-10 regulate lipopolysaccharide-stimulated human choriodecidual cytokine and prostaglandin E2 production. J Immunol 2003;170:158–66. [15] Malek A, Sager R, Schneider H. Effect of hypoxia, oxidative stress and lipopolysaccharides on the release of prostaglandins and cytokines from human term placental explants. Placenta 2001;22(Suppl. A):S45–50. [16] Ejima K, Koji T, Tsuruta D, Nanri H, Kashimura M, Ikeda M. Induction of apoptosis in placentas of pregnant mice exposed to lipopolysaccharides: possible involvement of Fas/Fas ligand system. Biol Reprod 2000;62:178–85. [17] Asagiri K, Nakatsuka M, Konishi H, et al. Involvement of peroxynitrite in LPS-induced apoptosis of trophoblasts. J Obstet Gynaecol Res 2003;29:49–55. [18] Duncan JR, Cock ML, Rees S, Harding R. The effects of repeated endotoxin exposure on placental structure in sheep. Placenta 2003;24:786–9. [19] Adamson SL, Whiteley KJ, Langille BL. Endothelin-1 constricts fetoplacental microcirculation and decreases fetal O2 consumption in sheep. Am J Physiol 1996;270:H16–23. [20] Wanecek M, Weitzberg E, Rudehill A, Oldner A. The endothelin system in septic and endotoxin shock. Eur J Pharmacol 2000;407:1– 15. [21] Battistini B, Forget MA, Laight D. Potential roles for endothelins in systemic inflammatory response syndrome with a particular relationship to cytokines. Shock 1996;5:167–83. [22] Lundberg JM, Ahlborg G, Hemsen A, et al. Evidence for release of endothelin-1 in pigs and humans. J Cardiovasc Pharmacol 1991;17 (Suppl.):350–3.
Proliferative responses in the placenta after endotoxin exposure in preterm fetal sheep§ Yves Garnier a,b,*, Mamed Kadyrov c, Markus Gantert a,b, Anke Einig a,c, Werner Rath a, Berthold Huppertz c,d a
Department of Obstetrics and Gynecology, University Hospital RWTH Aachen, Germany b Department of Obstetrics and Gynecology, University Hospital Cologne, Germany c Institute of Anatomy II, University Hospital RWTH Aachen, Germany d Institute of Cell Biology, Histology and Embryology, Medical University Graz, Austria
Received 2 August 2006; received in revised form 5 August 2007; accepted 22 August 2007
Abstract Objectives: Antenatal infections are associated with an increased risk of perinatal morbidity and mortality. Systemic application of endotoxins to the fetus results in an increase in placental vascular resistance and chronic reduction in umbilical blood flow. We studied morphological alterations of the placenta in response to fetal inflammation in the preterm sheep. Study design: Therefore, 14 fetal sheep were chronically instrumented at a mean gestational age of 107 1 days (term is 147 days). Four days after surgery fetuses received 100 ng lipopolysaccharide (LPS; n = 8) or saline (control; n = 6) intravenously. Fetal heart rate and arterial blood pressure were monitored continuously while blood gases and acid–base balance were measured at time points 0, +1, +3, +6, +12, +24, +48 and +72 h. Three days after LPS application placental cotyledons were analyzed by immunohistochemistry and morphometry. Different primary antibodies like AE 1 and AE 3 against cytokeratins were used. Secondary antibodies were visualized with 3-amino-9-ethylcarbazole (AEC) or using the Vectastain kit (Vector Laboratories, Burlingame, CA). Double staining was carried out first by utilizing Vectastain kit (black), followed by AEC staining (red). Counterstaining was performed with haematoxylin. Results: Fetal tachycardia and hypertension were induced transiently during the first 12 h after LPS application. Fetuses suffered from mild hypoxaemia while acidemia was absent. Morphometry revealed a non-significant shift in the relation of maternal and fetal placental compartments towards the maternal parts in response to LPS treatment. Endotoxin induced an increased proliferation in both compartments of the placenta with a 3.2-fold increase on the maternal and a 1.8-fold increase on the fetal side. Conclusions: Systemic endotoxin exposure of the preterm fetal sheep leads to a change in the gross organization of the placenta and changes in the proliferation patterns in both placental compartments. These rearrangements inside the placenta may disturb its organ function and subsequently lead to fetal morbidity associated with the fetal inflammatory response syndrome and chronic placental dysfunction, respectively. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Fetal inflammatory response syndrome; Chorioamnionitis
1. Introduction
§ The paper contains data that has been presented at, and awarded a prize by, the German Society for Gynaecology and Obstetrics in 2004. * Corresponding author at: University Hospital of Cologne, Department of Obstetrics and Gynecology, Kerpenerstrasse 34, D-50924 Cologne, Germany. Tel.: +49 221 478 4910; fax: +49 221 478 4929. E-mail address: [email protected] (Y. Garnier).
Clinical and epidemiological studies provide evidence that chorioamnionitis gives rise to a fetal inflammatory response. The histology of chorioamnionitis is characterized by polymorph nuclear infiltrations in the placenta and the fetal membranes, with its pathogenesis still unclear. An increasing amount of data supports the existence of asymptomatic bacterial colonialization already during the
0301-2115/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2007.08.016
Y. Garnier et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 152–157
first and second trimester of pregnancy. This silent inflammation may inhibit placental angiogenesis and thus modulate the course of the pregnancy in the long term. The progress of infection leads to a fetal systemic inflammatory response (FIRS), which histologically presents as funisitis (omphalovasculitis). When the chorioamnionitis induces a systemic inflammation in the pregnant woman, the clinical picture of an amnion infection syndrome (AIS) develops, including maternal fever and elevation of infection-related proteins in the maternal compartment. This inflammation contributes to neonatal disorders such as periventricular leukomalacia or bronchopulmonary dysplasia [1–6]. Yoon et al. have shown that antenatal exposure to intra-amniotic inflammation and documented fetal inflammatory response such as funisitis are strong and independent risk factors for the subsequent development of cerebral palsy at the age of 3 years [6]. Histologically, chorioamnionitis is defined by the presence of polymorphonuclear infiltrates in the placenta and its membranes. It affects 20% of term pregnancies and up to 60% of preterm pregnancies and is often an occult finding [7]. Grether and Nelson [4] reported that both clinical and histopathological evidence of placental infection were associated with an increased risk of unexplained cerebral palsy (CP) [OR 9.3, 95% CI 2.7–31 for clinical chorioamnionitis (CA); OR 8.9, 95% CI 1.9–40 for histological CA]. Recently, a meta-analysis confirmed the potential association between CA and CP, in both full term and preterm infants [8]. Lipopolysaccharides (LPS) which are released from gramnegative bacteria are known as potent inflammatory agents that have been previously used to induce a fetal inflammatory response. Data in experimental models demonstrate that application of LPS in premature fetal sheep affects fetal cardiovascular function in a dose-dependent manner [3]. Thus in preterm fetal sheep low dose endotoxin application caused a transient fall in umbilico-placental blood flow, dysregulation of cerebral blood flow and subsequent periventricular brain white matter injury [9–12]. The purpose of the present study was to study whether LPS administration as a paradigm for fetoplacental inflammation results in histopathological changes in the gross organization of the placenta in preterm fetal sheep. These observations may contribute to the understanding how the long-lasting cascades from endotoxin exposure to periventricular leukomalacia or bronchopulmonary dysplasia work out and may give a key function to the placenta.
2. Materials and methods 2.1. Animal preparation The protocols were approved by the Animal Medical Ethics Committee of Maastricht University and met the guidelines of the responsible governmental agencies.
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Fourteen fetal sheep were chronically instrumented at a mean gestational age of 107 1 days (term is 147 days) as described recently [9–11]. Briefly, all ewes underwent surgery with the use of sterile techniques while they were under general anesthesia (1 g thiopental sodium/70 kg intravenously for induction, 0.5–1.0% halothane in a 1:1 nitrous oxide and oxygen mixture for maintenance). The ewes received 1 g ampicilline (Pentrexyl1; Bristol-Myers, Woerden, The Netherlands) s.c. and 10 mg buprenorphin (Temgesic1; Schering, The Netherlands) per kg body weight twice a day for three consecutive postoperative days. A midline abdominal incision was performed. The fetal limbs were identified and exteriorized through an incision in the uterus. Polyvinyl catheters (Maxxim Medical BV, Den Bosch, The Netherlands) with 0.75 mm inner diameter and 1.25 mm outer diameter were inserted via a tibial fetal vein and artery of each hindleg and advanced into the caudal vena cava and abdominal aorta, respectively. The fetal skin was closed with cyanoacrylate glue (Cyanolit, Japan). An intrauterine pressure catheter was placed, and the uterus was sutured after replacing the lost amniotic fluid with a 0.9% saline solution at 39 8C. Catheters were filled with heparin (100 IU/ml; Heparin-Natrium1, Braun, Melsungen, Germany) and exteriorized through a small incision in the flank of the ewe. Catheters were protected by a pouch sewn to the skin of the ewe’s flank. A recovery period of 4 days followed the operation before experiments were started. Ewes were housed in individual cages and had free access to food and water. 2.2. Experimental protocol The experiments were performed 4 days after surgery at 111 1 days. The fetal inflammatory response was induced by endotoxin treatment (100 ng LPS (n = 8) i.v.; derived from E. coli; O127:B8, Sigma–Aldrich) while the control group (n = 6) received 2 ml 0.9% saline i.v. The total number of animals included in the experiment was 14. Blood samples were taken at various time points before (1 h) and after (+1, +3, +6, +12, +24, +48, +72 h) LPS injection from the fetal descending aorta and analyzed for blood gases, acid base balance (AVL 993, Radiometer, Copenhagen, Denmark), hemoglobin concentration and arterial oxygen saturation of hemoglobin (OSM 2 Hemoximeter, Radiometer, Copenhagen, Denmark). Three days after endotoxin administration the animals were anesthetized with sodium pentobarbitone (1 g/70 kg intravenously for induction, 0.5–1.0% halothane in a 1:1 nitrous oxide and oxygen mixture for maintenance). The right atrium and both jugular veins were incised. After heparin infusion (10.000 IU), each animal was perfusionfixed through a needle placed in the left ventricle with 500 ml of buffered paraformaldehyde (4%) until the fluid efflux from the jugular veins was clear. Ewes and fetuses were killed by a maternal overdose of sodium pentobarbitone (150 mg/kg). Placentomes including the maternal
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caruncle and the fetal cotyledon were weighed and fixed for 24 h in phosphate-buffered formaldehyde (4%). Then the specimens (one half of a placentome, from the maternal to the fetal side) were paraffin-embedded. Standard hematoxylin and eosin stains were used to assess histological structures. 2.3. Immunohistochemistry Routine immunohistochemical procedures were performed on serial sections of 5 mm thickness with anticytokeratin type I (used as epithelial marker; dilution 1:100; clone AE1; Serotec). Other antibodies directed against cytokeratins 7, 18, 19, or other pan cytokeratin antibodies such as clone MNF 116 did not reveal any reliable staining. Anti-Ki-67 was used as a proliferation marker (dilution 1:30; clone Mib-1; Dako). Binding of species-specific biotinylated secondary antibodies was visualized with 3amino-9-ethylcarbazole (AEC). For quantification of cell numbers double staining was performed with anti-Ki-67 first utilizing the Vectastain kit (Vector Laboratories, Burlingame, CA) (black) and then anti-cytokeratin type I with AEC (red). Sections were counterstained with haematoxylin. Negative control reactions omitting the primary antibodies or using an IgG1-negative control antibody (DAK-GO1; Dako) displayed no staining.
2.5. Statistical analysis 2.5.1. Physiologic variables and blood gas parameters Statistical analysis was performed by Super Anova Statistical Package (Abacus, Inc., CA, USA). Games– Howell-test was used as post hoc testing procedure. Statistical significance was set at p < 0.05. All data are expressed as mean values S.D. 2.5.2. Histology Placental cellular proliferation and the amount of fetal versus maternal tissues were counted by an investigator (MK) blinded to the experimental protocol. Numerical density and rates of Mib-1 positive cells were calculated for every single section. Then mean and S.D. were calculated for every single section and then for every placenta before calculating mean and S.D. for each group and each investigated variable. Comparisons between groups were carried out using one-way analysis of variance (ANOVA) and post hoc Bonferroni’s t-test. Statistical significance was set at p < 0.05.
3. Results 3.1. Fetal heart rate, blood pressure, blood gases and acid–base balance
2.4. Quantification Measurements were performed on a stereological workstation (Olympus CAST grid; Olympus, Albertslund, Denmark; Olympus BX 50 microscope), and sections were visualized via a CCD color video camera (600 800 pixels; JAI, Glostrup, Denmark) on a 17 in. monitor. On sections stained with anti-cytokeratin type I we were able to differentiate between maternal and fetal tissues. Using a low magnification (lens: 1.25; PlanApo) the area of interest covering the whole placentome was delineated. Using systematic random sampling a computer program selected the counting fields within the area of interest. Fifty-five to 150 fields (depending on the size of the area of interest) were evaluated per section (lens: 20; UPlanApo, NA = 0.70) by taking a grid with 16 crosses and counting the number of crosses per fetal or maternal tissue. On serial sections double-stained with anti-cytokeratin type I and Mib-1, the ‘areas of interest’ were delineated again, covering the whole placentome. Again, the computer chose the counting fields within the area of interest. Then the number of Mib-1 positive maternal and fetal cells (lens: 20; UPlanApo, NA = 0.70) was counted in 55–150 fields per section. Field sizes were determined and the numerical density of Mib-1 positive cells was calculated as cell profiles per area of the maternal and fetal tissues. Sections from one to four paraffin blocks per placentome were analyzed.
All measurements of FHR, MAP, blood gases and acid base balance during the control period were in the normal range for chronically prepared fetal sheep of this gestational age [9–11]. There were no significant differences between the control and LPS group at this time point (Table 1). MAP in the control group was 41 3 mmHg during normoxia, and did not significantly change during the course of the experiment. In response to LPS exposure MAP increased at +1 h (+21%; p < 0.001) and returned to control levels 4–5 h thereafter. FHR increased 3 and 6 h after LPS injection (+10%, p < 0.05; and +16%, p < 0.01), and was significantly higher than FHR in control animals ( p < 0.01) which did not change significantly from baseline. FHR normalized within 24 h after LPS injection. Oxygen saturation of fetal hemoglobin was transiently reduced in response to endotoxaemia (time point +6 h: 41%; p < 0.05) and slowly recovered during the following 24 h although not completely ( p < 0.05 at 72 h). There were no significant differences in arterial blood pH and blood gas parameters between control and LPS treated animals (Table 1). 3.2. Histology Control animals showed no structural abnormalities or signs of inflammation in both the fetal and maternal compartments of the placenta. LPS treatment caused an increase in proliferation in both compartments, which was visualized by double immunohistochemical staining
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Table 1 Fetal heart rate, mean arterial pressure, acid–base balance and blood gases in control (n = 6) and LPS (E) treated (n = 8) preterm fetal sheep Group
0 h
+1 h
+3 h
+6 h
+12 h
+24 h
+48 h
+72 h
Fetal heart rate (beats per min) C 176 6 172 10 E 179 8 169 12
184 12 193 13y
185 12 207 9§,y
184 10 205 12§,y
171 9 181 8
186 9 177 9
185 13 193 14§,y
Mean arterial blood pressure (mmHg) C 41 3 41 3 E 41 3 46 5§,y
43 3 42 4
43 5 39 4
43 4 39 5
40 4 37 4
45 4 39 5
45 4 34 5y
Arterial oxygen saturation C 49.3 5.6 E 52.5 8.7
51.6 3.4 53.2 7.4
52.8 6.8 41.0 7.3
55.5 5.8 31.2 6.8§,y
48.8 6.3 42.2 7.9
54.3 5.5 46.6 4.9
53.6 12.6 50.5 5.6
56.2 11.9 38.8 9.2§,y
Hemoglobin (mg/dl) C 10.1 0.3 E 10.3 0.5
10.5 0.6 10.9 0.8
10.6 0.8 11.8 1.2§
10.3 0.5 12.5 1.1
10.4 0.6 11.4 1.1
10.0 1.1 10.1 0.9
10.3 0.7 10.3 0.6
10.5 0.5 10.4 1.0
PO2 (mmHg) C 22.0 2.6 E 22.7 2.1
21.8 2.7 23.1 2.9
20.9 2.7 21.0 2.6
22.8 2.4 17.3 3.2
21.9 2.0 20.0 2.9
24.2 1.8 21.5 3.0
24.2 3.4 22.7 3.0
22.7 2.9 22.0 2.9
PCO2 (mmHg) C 54.6 3.0 E 54.0 2.9
56.8 5.7 55.7 2.7
56.5 5.4 59.8 4.8
59.3 5.3 62.9 6.0
47.8 6.2 62.0 6.8
53.0 4.2 55.9 5.8
56.2 4.3 52.0 6.7
62.0 5.0 61.1 8.2
pH C E
7.36 0.02 7.33 0.02
7.37 0.04 7.32 0.05
7.35 0.04 7.32 0.04
7.35 0.02 7.33 0.06
7.34 0.01 7.33 0.04
7.36 0.03 7.31 0.06
7.34 0.04 7.32 0.06
7.35 0.03 7.37 0.04
Values are given as means S.D. § p < 0.01 significant between groups. y p < 0.01 significant vs. control within groups.
(Fig. 1). Immunohistochemical staining using antibodies against cytokeratins revealed a clear staining of the fetal trophoblast with a much weaker staining of the remaining maternal uterine epithelial compartment (Fig. 1). This enabled us to differentiate between both the fetal (delineated by trophoblast) and the maternal compartments. After LPS treatment (Fig. 1B) a clear increase in the number of Mib-1 positive nuclei became obvious compared to control tissues (Fig. 1A). Quantification of Mib-1 positive nuclei revealed a significant increase of positive cells in the maternal (control: 0.225 0.095 cells/field; LPS: 0.730 0.259) as well as in the fetal (control: 2.425 0.418 cells/ field; LPS: 4.400 1.224) compartment (Fig. 2A). In the maternal part of the placenta the level of proliferation
increased 3.2-fold, while in the fetal part the increase was only 1.8-fold (Fig. 2A). At the same time the relation between the volumes of maternal and fetal tissues did not change significantly (Fig. 2B). In control tissues the relative distribution of maternal to fetal tissues was 1.55 0.47, and in LPS treated tissues the relative distribution was 1.43 0.27.
4. Discussion In the present study we have shown that intrauterine exposure of the fetus to endotoxaemia, induced by intravenously applied LPS, results in a fetal inflammatory
Fig. 1. Double immunohistochemistry for cytokeratin and Mib-1. (A) In placental tissues from control animals a normal distribution of proliferating cells in the maternal (M) and fetal (F) compartments of the placenta can be seen. (B) In placental tissues from animals treated with 100 ng LPS an increased proliferation level becomes obvious, both in the maternal (M) as well as in the fetal (F) compartment. Magnification 800.
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Fig. 2. Quantification of changes due to LPS treatment. (A) Quantification of the level of proliferation revealed a 3.2-fold increase in the proliferation within the maternal compartment after treatment with LPS. In the fetal compartment this increase was only 1.8-fold. (B) The relative distribution of maternal and fetal tissues does not change significantly after LPS treatment.
response syndrome, i.e. mild and transient depression of fetal cardiovascular function, e.g. fetal tachycardia and hypertension and mild hypoxaemia. These changes occurred transiently and fetal cardiovascular function completely recovered within 72 h after endotoxaemia was induced. Previously, we have shown that under such circumstances an increase in placental vascular resistance and a reduction in umbilical blood flow are present [9,10]. Moreover, this insult led to an inflammatory response in the fetal brain, with cystic lesions in the periventricular white matter, which are often present in immature fetuses with documented chorioamnionitis or funisitis [11,12]. In the present study low dose LPS administration caused a fetal and placental inflammation, which is common under clinical conditions. It is important to note, that the chronic fetal inflammatory response observed in the present study has been induced by a single-shot dose of endotoxin. Although we have not measured endotoxin plasma levels these have been estimated by concerning the fetal blood volume, plasma volume of the fetal blood volume and the administered LPS doses. Thus, the median endotoxin plasma concentrations were within the range observed in patients
with clinical sepsis [13]. In this context it has to be mentioned that humans are much more sensitive to endotoxin than other species, including the sheep. Moreover, the endotoxic effect of LPS widely varies with the bacterial strain from which it has been isolated. This is the first study to show that fetal endotoxaemia caused an increased proliferative response in both compartments of the sheep placenta. Using morphometry and double immunohistochemistry both the relation between fetal and maternal tissues and the proliferation index within both compartments has been quantified. Using immunohistochemistry stainings of the placenta the present study demonstrates that the three epithelial parts of the sheep placenta, i.e. uninucleate trophoblast cells, binucleate cells and all derivatives of the uterine epithelium can be detected separately. Morphologic changes in the placenta in response to LPS treatment might be induced by different mechanisms. In studies using human placental explants LPS resulted in an increase in the release of prostaglandins (PGF2alpha) and proinflammatory cytokines such as tumor necrosis factoralpha, interleukin (IL)-1beta and IL-10 [14]. These responses were similar to that seen after severe oxidative stress in this model [15]. Oxidative stress, which generates reactive oxygen species (ROS), has been postulated to be involved in the pathophysiology of some complications such as infectious disease and preeclampsia during pregnancy, occasionally leading to placental dysfunction or abruptio placentae. Under these circumstances proliferation and apoptosis are increased in the placenta [15–17]. However, differences of the anatomic settings between the sheep and the human placenta have to be taken into account. While there is a haemochorial placental type in the human, the sheep placenta is of the epitheliochorial type. Changes in the fetal compartment of the placenta should arise irrespective of the type of placentation. As to the changes in the maternal compartment, in both settings any factor has to cross the trophoblast layer to reach the maternal compartment. If there is a connective tissue behind this layer as in the sheep it may result in a local effect. If there is blood as in the human, this may lead to a more systemic response in the human. Duncan et al. have demonstrated that repeated exposure to endotoxin in immature fetal sheep reduced both the placental weight and the average cross-sectional area of placentomes [18]. In this context it is important to note that endotoxin exposure of the immature fetal sheep causes a substantial and long-lasting decrease in umbilico-placental blood flow resulting in mild to sustained fetal hypoxaemia, depending on the protocol used [3,9,10]. It has been reported that placental blood flow began to fall 1 h after LPS and was lowest (40%) at 4–5 h after LPS, while placental vascular resistance rose by 75% during this period [9]. Thereafter, placental blood flow slowly returned to control values at 12– 16 h after endotoxaemia was induced. The increase in umbilico-placental vascular resistance during endotoxaemia might be mediated by endothelin (ET-1). ET-1 is a potent
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endothelium-derived vasoconstrictive peptide that is able to constrict fetoplacental microcirculation and decrease fetal oxygen consumption in fetal sheep [9,19]. It has been shown previously, that ET-1 is released in response to several stimuli, including hypoxia and endotoxaemia [20]. Among the pathophysiologic conditions known to involve the endothelin system, sepsis and endotoxaemia are producing the highest plasma levels of ET-1 [21]. In the present study we did not measure endothelin serum levels, due to the lack of specificity of the test sera and the instability of the molecule in the laboratory setting. However, bacterial endotoxin and septic conditions are known to increase ET-1 concentrations in fetal umbilical arterial plasma more than fivefold in both pigs and humans, reaching levels close to threshold vasoconstriction [22], while the placental microcirculation is remarkably inert to many vasoconstrictors, including norepinephrine and angiotensin II. Thus we speculate, that ET-1 has a critical role in the control of placental microcirculation and might be the mediator of transitory placental hypoperfusion during endotoxaemia seen in the present study. Moreover, other proinflammatory agents such as IL-1, TNF-alpha and TGF-beta, which are released during endotoxaemia, also increase the production of ET-1 from endothelial cells. This placental inflammatory response might have an impact in the pathogenesis of various pregnancy complications such as preterm labor, preeclampsia, abruptio placentae and intrauterine growth restriction, respectively.
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