Plasma clusterin increased prior to small for gestational age (SGA) associated with preeclampsia and decreased prior to SGA in normotensive pregnancies.

In our search for early biomarkers for the pregnancy complicationssmall for gestational age (SGA) and preeclampsia (PE) we analysed plasma from 19-21 weeks gestation in women recruited into the SCOPE study, a prospective cohort of nulliparous women, by differential in gel electrophoresis (DIGE). DIGE revealed the differential expression of clusterin levels and its isoforms in top6-depleted plasma of women who delivered an SGA infant but remained normotensive (SGA-NT; N = 8) compared to healthy women with an uncomplicated pregnancy outcome (Controls, N = 8). Immunosorbent enzyme-linked assay (ELISA) showed that compared to plasma clusterin levels from healthy controls [71.1 (SD 12.4) µg/mL, n = 39], clusterin was decreased in SGA-NT [58.3 (SD 11.7), N = 20, P < 0.0001], increased in women with SGA and PE [81.5 (SD 14.8), N = 20, P < 0.01], but similar in PE alone [71.2 (SD 9.4)g/ml, P = 1.0]. Screening for clusterin levels and/or its different isoformsmay be useful in mid-pregnancy to identify women who subsequently develop SGA but remain normotensive or who develop preeclampsia with SGA.

Pre-eclampsia is associated with elevated CXCL12 levels in placental syncytiotrophoblasts and maternal blood.

OBJECTIVES: Placental derived vasculogenic/angiogenic substances in maternal blood are dysregulated in pre-eclampsia. We hypothesized that CXCL12, a chemokine with vasculogenic actions, is amongst such molecules. STUDY DESIGN: CXCL12, CXCL16, CXCR4, and CXCR6 immunolocalization in placental tissue was analyzed in pre-eclampsia (n=8) in comparison to controls (n=8). CXCL12, measured by ELISA in blood, in women diagnosed with pre-eclampsia (n=14) and prior to the development of pre-eclampsia (at 20 weeks' gestation, n=20) was compared with CXCL12 concentrations in gestation-matched, healthy control subjects (n=34). RESULTS: In placental tissue, syncytiotrophoblast staining for CXCL12 was increased in pre-eclampsia. Maternal serum CXCL12 was increased in pre-eclampsia [2000 (SD 402) vs 1484 (SD 261)pg/ml, P=0.01] but not in plasma obtained at 20 weeks of gestation prior to the onset of pre-eclampsia [1183 (SD 336) vs 1036 (SD 144)pg/ml, P=0.09]. CONCLUSION: Our data suggest that the syncytiotrophoblast contributes to a pre-eclampsia-associated increase in CXCL12 levels in maternal blood. These findings support the hypothesis that an imbalance of angiogenic factors contributes to the pathogenesis of pre-eclampsia.

Aberrant processing of plasma vitronectin and high-molecular-weight kininogen precedes the onset of preeclampsia.

To date, there is no reliable test to identify women in early pregnancy at risk of developing preeclampsia. Difference gel electrophoresis (DIGE) identified the plasma proteins vitronectin (VN) and high-molecular-weight kininogen (HK) in association with preeclampsia. In a longitudinal proteomics study, the plasma of preeclamptic patients (n = 6) was compared to healthy control participants (n = 6) before the onset of preeclampsia (week 20) and at the time of presentation with clinical disease (weeks 33-36). The 75-kd single-chain VN molecule increased 1.6- to 1.9-fold in preeclampsia, whereas the 65-kd moiety of the 2-chain VN molecule decreased 1.5- to 1.7-fold compared to healthy controls (P < .05). Immunoblots revealed differences in proteolytic processing of VN and/or HK in women who develop preeclampsia or preeclampsia further complicated by small-for-gestational-age. Vitronectin and HK may prove to be useful as early markers of fibrinolytic activity and neutrophil activation, which are known to be associated with preeclampsia.

A proteomic approach identifies early pregnancy biomarkers for preeclampsia: novel linkages between a predisposition to preeclampsia and cardiovascular disease.

Preeclampsia (PE) is a common, potentially life-threatening pregnancy syndrome triggered by placental factors released into the maternal circulation, resulting in maternal vascular dysfunction along with activated inflammation and coagulation. Currently there is no screening test for PE. We sought to identify differentially expressed plasma proteins in women who subsequently develop PE that may perform as predictive biomarkers. In seven DIGE experiments, we compared the plasma proteome at 20 wk gestation in women who later developed PE with an appropriate birth weight for gestational age baby (n=27) or a small for gestational age baby (n=12) to healthy controls with uncomplicated pregnancies (n=57). Of the 49 differentially expressed spots associated with PE-appropriate for gestational age, PE-small for gestational age or both (p<0.05, false discovery rate corrected), 39 were identified by LC-MS/MS. Two protein clusters that accurately (>90%) classified women at risk of developing PE were identified. Immunoblots confirmed the overexpression of fibrinogen gamma chain and alpha-1-antichymotrypsin in plasma prior to PE. The proteins identified are involved in lipid metabolism, coagulation, complement regulation, extracellular matrix remodeling, protease inhibitor activity and acute-phase responses, indicating novel synergism between pathways involved in the pathogenesis of PE. Our findings are remarkably similar to recently identified proteins complexed to high-density lipoprotein and linked to cardiovascular disease.

An altered pattern of circulating apolipoprotein E3 isoforms is implicated in preeclampsia.

Preeclampsia is a common pregnancy complication that is an important cause of preterm birth and fetal growth restriction. Because there is no diagnostic test yet available for preeclampsia, we used a proteomic approach to identify novel serum/plasma biomarkers for this condition. We conducted case control studies comparing nulliparous women who developed preeclampsia at 36-38 weeks of gestation with healthy nulliparous women matched by gestational age at sampling. Serum/plasma was depleted of six abundant proteins and analyzed by two-dimensional gel electrophoresis (n = 12 per group) and difference gel electrophoresis (n = 12 per group). Differences in abundance of protein spots were detected by univariate and multivariate statistical analyses. Proteins were identified by mass spectrometry and expression of selected proteins was validated by immunoblotting. Proteins whose concentrations were selectively associated with preeclampsia included apolipoprotein E (apoE), apoC-II, complement factor C3c, fibrinogen, transthyretin, and complement factor H-related protein 2. An increase in a deglycosylated isoform of apoE3 and concomitantly decreased amounts of one apoE3 glycoisoform were identified in preeclamptic plasma and confirmed by immunoblotting. Altered production of these preeclampsia-related apoE3 isoforms might impair reverse cholesterol transport, contributing to arterial damage. These findings point to a novel mechanistic link between preeclampsia and subsequent cardiovascular disease.

Identification of suppressors of cytokine signaling (SOCS) proteins in human gestational tissues: differential regulation is associated with the onset of labor.

Inflammatory cytokines secreted by the placenta and fetal membranes are believed to play an important role in the initiation of parturition. The suppressor of cytokine signaling (SOCS) proteins regulate signal transduction by several cytokines that have been reported to affect gestational tissues. The presence, distribution and roles of SOCS proteins, however, have not been described in human gestational tissues. Using reverse transcriptase (RT)-PCR and Western blot analysis we investigated the expression of SOCS1, SOCS2, and SOCS3 mRNA and protein, respectively, by human villous placenta, amnion and choriodecidua (n = 3-4). Tissues were obtained from uncomplicated pregnancies at term after either spontaneous labor and vaginal delivery or caesarean section (before labor). Messenger RNAs for SOCS1, SOCS2, and SOCS3 were expressed in all tissue types, irrespective of labor status. SOCS proteins were, however, only detectable in villous placenta and in one case in the choriodecidua. Labor was associated with abrogated expression of SOCS1 and SOCS3 proteins in villous placenta and the choriodecidua sample. Following labor the band for SOCS2 protein increased slightly in size which may indicate post-translational modification of SOCS2. Reduced expression of SOCS proteins in gestational tissues may provide a mechanism by which inflammatory cytokines enter into a positive feedback loop of inflammatory changes leading to delivery.

UDP-glucuronosyltransferase activity, expression and cellular localization in human placenta at term.

The activity, expression and localization of the UDP-glucuronosyltransferases (UGTs) were investigated in human placenta at term. UGT activity (measured with the substrate 4-methylumbelliferone (4-MU)) was observed in all 25 placentas sampled and maximum velocity (V(max)) ranged 13-fold from 5.1+/-0.9 to 66.9+/-17.5 nmol/min/mg protein (mean+/-SD). Substrate affinity (K(m)) ranged 5-fold from 246+/-24 to 1124+/-422 microM. Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of the isoforms UGT2B4, 2B7, 2B10, 2B11 and 2B15 was observed in all (12/12) placentas sampled and expression of UGT2B17 was noted in 8/12 placentas. Northern analysis of the UGT2B7 isoform in 12 placentas revealed a 10-fold difference in expression with RT-PCR variability and the 13-fold variation observed in UGT activity. The presence of UGT2B4 and 2B7 proteins (52 and 56kDa, respectively) was demonstrated by Western blotting. The sites of placental UGT2B transcription (in situ hybridization) and protein expression (immunohistochemistry) were located in the syncytium of the placental trophoblasts bordering the placental villi. UGT1A proteins could not be observed with immunohistochemistry or Western blotting and expression could not be observed with RT-PCR. Our discovery of UGT expression and activity at the site of maternal-fetal exchange is consistent with a role for UGTs in detoxification of exogenous and endogenous ligands and the maintenance of placental function through clearance and regulation of steroid hormones.