Steven R. Allen

Ultrasonographic diagnosis of congenital anomalies in twins

To determine whether serial ultrasonographic examinations with basic anatomic surveys provide an adequate screen for congenital abnormalities that are more common in twins, we compared the results of prenatal sonograms and neonatal examinations for 314 twins (157 pairs) delivered during a recent 42-month period. An anomaly was defined as major if it potentially required surgical repair or precluded normal life expectancy; otherwise it was defined as minor. Thirty-three twins (9.5%) had 40 anomalies; 28 (9%) were major and 12 (4%) were minor. Prenatal ultrasonography with cardiac screening limited to the four-chamber view provided detection of 39% of all major anomalies, 55% of noncardiac major anomalies but none of the cardiac lesions, and 69% of the major anomalies for which routine prenatal management should be altered. No false-positive diagnoses incorrectly altered management. We conclude that serial prenatal ultrasonographic examinations are useful in detecting noncardiac anomalies for which twins are at increased risk, but the four-chamber view is not an adequate screen for the cardiac malformations of twins.

Hyperglycemia impairs cytotrophoblast function via stress signaling

OBJECTIVE Diabetes mellitus is a risk factor for preeclampsia. Cytotrophoblast (CTB) invasion is facilitated from the conversion of plasminogen to plasmin by urokinase plasminogen activator (uPA), regulated by plasminogen activator inhibitor 1 (PAI-1), and may be inhibited in preeclampsia. This study assessed signaling mechanisms of hyperglycemia-induced CTB dysfunction. STUDY DESIGN Human CTBs were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 hours. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligand (rosiglitazone). Expression of uPA, PAI-1, and PPAR-γ levels and p38 mitogen-activated protein kinase phosphorylation were measured by Western blot in cell lysates. Messenger ribonucleic acid of uPA and PAI-1 was measured by quantitative polymerase chain reaction. Levels of interleukin-6, angiogenic (vascular endothelial growth factor [VEGF], placenta growth factor [PlGF]) and antiangiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], soluble endoglin [sEng]) were measured in the media by enzyme-linked immunosorbent assay kits. Statistical comparisons were performed using analysis of variance with a Duncans post-hoc test. RESULTS Both uPA and PAI-1 protein and messenger ribonucleic acid were down-regulated (P < .05) in CTBs treated with 135 mg/dL glucose or greater compared with basal (45 mg/dL). The sEng, sFlt-1, and interleukin-6 were up-regulated, whereas the VEGF and PlGF were down-regulated by 135 mg/dL glucose or greater. p38 phosphorylation and PPAR-γ were up-regulated (P < .05) in hyperglycemia-treated CTBs. The SB203580 or rosiglitazone pretreatment showed an attenuation of glucose-induced down-regulation of uPA and PAI-1. CONCLUSION Hyperglycemia disrupts the invasive profile of CTB by decreasing uPA and PAI-1 expression; down-regulating VEGF and PlGF; and up-regulating sEng, sFlt-1, and interleukin-6. Attenuation of CTB dysfunction by SB203580 or rosiglitazone pretreatment suggests the involvement of stress signaling.

A p38 mitogen-activated protein kinase inhibitor attenuates cardiotonic steroids-induced apoptotic and stress signaling in a Sw-71 cytotrophoblast cell line

INTRODUCTION Preeclampsia (preE) is characterized by abnormal placentation. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits the function of cytotrophoblast cells (CTBs). We demonstrated that CTSs induce anti-angiogenic and anti-proliferative effects in Sw-71 CTBs. This study tests that CTSs induce apoptotic and stress signaling. METHODS Human extravillous Sw-71 CTBs were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Some cells were pretreated with 10 μM p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) for 2 h prior to CTSs treatment. We analyzed p38 MAPK phosphorylation, expression of pro-inflammatory protein cyclooxygenase-2 (Cox-2) and ratio of pro-apoptotic Bcl-2-associated X protein (Bax) to anti-apoptotic Bcl-2 protein by western blot in CTSs-treated CTBs lysates. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncans post hoc test. RESULTS p38 MAPK phosphorylation, expression of Cox-2 and Bax/Bcl-2 was upregulated (*p < 0.05) in CTBs exposed to ≥ 0.1 nM CTSs. Secretion of sFlt-1 and sEng were increased while VEGF and PIGF were decreased in Sw-71 CTBs treated ≥1 nM of each CTSs (*p < 0.01 for each). The SB203580 pretreatment of CTBs significantly attenuated CTS-induced effects. DISCUSSION Exposure of Sw-71 CTBs to CTSs induced apoptotic and stress signaling and causing anti-angiongenic effect. The observed diminution of CTS-induced signaling by SB203580 pretreatment implicates p38 MAPK as a regulator of these pathways.

Attenuation of hyperglycemia-induced apoptotic signaling and anti-angiogenic milieu in cultured cytotrophoblast cells

ABSTRACT Objective: Preeclampsia (preE) is a hypertensive disorder that occurs 20% in diabetic pregnancy. We have shown that hyperglycemia impairs cytotrophoblast cell (CTB) function. In this study, we assess apoptotic and anti-angiogenic signaling in excess glucose-induced CTBs. Study Design: Human extravillous CTBs (Sw. 71) were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor gamma (PPARγ) ligand (rosiglitazone) or with D-mannitol. Cell lysates were utilized to measure p38 MAPK phosphorylation, PPARγ, Bcl-2-associated-X protein (Bax), anti-apoptotic Bcl-2, caspase-9, and cyclooxygenase-2 (Cox-2) expression by western blot. Levels of the vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), and interleukin 6 (IL-6) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: p38 phosphorylation and PPARγ were upregulated (p < 0.05) in CTBs treated with ≥150 mg/dL glucose compared to basal (100 mg/dL). Expressions of Bax/Bcl-2, Cox-2, and caspase-9 were upregulated (p < 0.05) in CTBs treated with ≥150 mg/dL glucose. Secretion of sFlt-1, sEng, and IL-6 was increased while VEGF and PIGF were decreased in CTB-treated ≥150 mg/dl of glucose (*p < 0.01 for each). SB203580 or rosiglitazone pretreatment significantly attenuated hyperglycemia-induced apoptotic and anti-angiogenic signaling. D-Mannitol had no effect. Conclusion: Hyperglycemia induced apoptotic and anti-angiogenic signaling in CTBs. The observed diminution of hyperglycemia-induced signaling by SB203580 or rosiglitazone pretreatment suggests the involvement of apoptotic and anti-angiogenic signaling in CTB dysfunction.

ID: 105: COMPARISON OF PLACENTAL AND PLASMA SOLUBLE (PRO)RENIN RECEPTOR IN NORMAL AND PREECLAMPTIC PREGNANCY

Objective Preeclampsia (preE), a syndrome of hypertension and proteinuria. Most recently it was demonstrated that high circulating levels of soluble (pro) renin receptor s(P)RR at delivery were associated with preE. In this study the placental expression of (P)RR were evaluated in preE patients and in a rat model of preE as well as in nonhuman primates. We also evaluated the circulatory levels of s(P)RR. Study Design (1) Placental samples were collected from 20 NP and 20 preE consenting patients in an IRB approved prospective study. (2) An established rat model of preE and NP rats (n=10 each) were used. (3) The placental samples from squirrel monkey (NP; n=10) and owl monkey (both early and term, NP, n=1) were collected. The (P)RR expression were measured both by western blotting (WB) and Immunohistochemistry (IHC) using anti-ATP6IP2. The levels of serum s(P)RR were measured by ELISA. Results The placental expression of (P)RR were higher (p<0.05) in preE compared to NP both in patients and rat model. The s(P)RR levels were higher in preE (preE patients: 29.2±4.5; PDS rats: 16.9±1.9 ng/mL) compared to NP (NP human: 19.3±4.2; NP rats: 10.4±3.7 ng/mL). The early placenta of owl monkey expressed higher (P)RR compared to term and were expressed in squirrel monkey placentas. Conclusions These data suggest that increased expression of (P)RR in the placenta are related to the occurrence of preE in both patients and rat models. These data also reconfirmed that the high level of circulatory s(P)RR is associated with preE. The higher expression of (P)RR in early owl monkey in compare to term placenta suggests that the (P)RR is important for normal placental development. The expression of (P)RR in nonhuman primates reveals the approach of future studies on owl monkey and squirrel monkey preE models.

ID: 56: COMPARISON OF OUTCOMES BETWEEN NORMAL AND PREECLAMPTIC PREGNANCIES: A PROSPECTIVE STUDY

Objective Preeclampsia (preE) is a multifaceted complication found uniquely in the pregnant patient and one that has puzzled scientist for years. It has been demonstrated that preE is not a single disorder, but a complex syndrome that is produced by various pathophysiologic triggers and mechanisms affecting 3–8% of obstetrical patients worldwide. PreE, is a major cause of premature delivery and maternal and fetal death. It is characterized by de novo development of hypertension and proteinuria after 20 weeks gestation. preE has a significant link to alterations of placental function leading to stress and apoptotic signaling, which pass the placental barrier and leave persistent defect in the circulation of the offspring. We assessed the comparison of pregnancy outcome between patients with and without preE. Methods We recruited 20 normal pregnant (NP) and 20 preE consenting patients after deliveries in an IRB approved prospective study from Scott & White Healthcare. We evaluated the following parameters for mothers BP, Proteinuria, BMI, Gestational age, Age, Placental factors: circumference, placental signaling proteins. The placental stress signaling proteins (p38 MAPK, COX-2 and Bax/Bcl-2) were measured. We also evaluated babies for IUGR and anthropometric measurements. Comparisons were performed using Students t test. Results Mothers in the PE group had significantly higher blood pressures (SBP p=0.0000001 and DBP p=0.001) and also higher urinary protein excretion (p=0.002). Average hospital stay for PreE babies were longer than NP babies (p=0.001879). No complications were reported for NP babies; however, preE babies had multiple complications like hypoglycemia, RDS etc though they were born at preterm. Many of the PreE babies were born premature (p=0.017). The birth weights of the PE babies were much lower than the NP babies with a p value of 0.027 and also the PE babies were significantly SGA when compare to the NP babies with significant difference in their Ponderal Index (PI) (p=0.0004). The placental stress signaling proteins p38 MAPK, COX-2 and Bax/Bcl-2 were up-regulated in preE compared to normal pregnancy (p<0.05, in each case). Conclusions PreE alters the intrauterine environment and activates the detrimental signaling that is transported to fetus resulting in premature deliveries, IUGR babies and their related complications like extended hospitalization.

ID: 71: PRAVASTATIN PROTECTS A SW-71 CYTOTROPHOBLAST CELL LINE FROM A HYPERGLYCEMIA-INDUCED PREECLAMPSIA PHENOTYPE

Objective An increasing level of evidence supports the utility of pravastatin as prevention against preeclampsia (preE). We previously demonstrated a hyperglycemia induced cytotrophoblast (CTBs) dysfunction characteristic of a preE-like phenotype and sought to demonstrate the utility of pravastatin in rescuing CTBs from this hyperglycemia induced dysfunction. Methods Human CTBs were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 hrs. Cells were treated with pravastatin (1 ug/mL) either alongside or 2 hrs prior to glucose exposure. Some cells were treated with D-Mannitol as a negative control for glucose exposure. Cell migration was performed by Matrigel migration assay kit according to manufacturer protocol. Cell lysates were utilized to evaluate the mRNA expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1), while also assessing proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng) and interleukin 6 (IL-6) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncans post hoc test. Results Hyperglycemia inhibited CTBs migration by down-regulating uPA, PAI-1, PCNA and up-regulating p38 phosphorylation in cells treated with >150 mg/dL glucose compared to basal (100 mg/dL) (*p<0.05 for each). Secretion of sFlt-1, sEng and IL-6 were increased while VEGF and PIGF were decreased in CTBs treated ≥150 mg/dl of glucose (*p<0.05 for each). Both pravastatin pretreatment and co-treatment significantly rescued CTBs migration, up-regulating uPA, PAI-1, PCNA, down-regulating p38 phosphorylation, and correcting the angiogenic profile of CTBs (p<0.05 for each). D-Mannitol showed no osmotic effect on CTBs. Conclusions Pravastatin mitigates the hyperglycemia-induced dysfunction of CTBs by attenuating the glucose-induced anti-proliferative, anti-migratory, anti-invasive and anti-angiogenic phenotype similar to that seen in Preeclampsia. This study supports the potential for pravastatin use on CTBs development early in pregnancy and the importance of continuing research of pravastatin in preE prevention.