Role of SOX9 and Hif-1α expression in placentas of patients with HELLP.

PURPOSE: In this study, we investigated the immunohistochemical staining of SRY-box transcription factor 9 (SOX9) and Hif-1α expression in placentas of pregnant woman with hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome. METHODS: Placentas of 20 normotensive and 20 women with HELLP syndrome were processed for routine histological tissue processing. The biochemical and clinical parameters of patients were recorded. Placentas were stained with hematoxylin-eosin and SOX9 and Hif-1α immunostaining. RESULTS: Normotensive placentas showed normal histology of placenta, however placentas of HELLP syndrome showed intense thrombosis, thinning of the villi membrane and vascular dilatation. In placentas of normotensive patients, SOX9 reaction was immunohistochemically negative, however placentas of HELLP group showed SOX9 expression in decidual cells, and syncytial regions of floating villi and inflammatory cells. In placentas of normotensive patients, Hif-1α reaction was mainly negative in vessels and connective tissue cells. Placentas of HELLP group showed increased Hif-1α expression in decidual cell and especially inflammatory cells in the maternal region. CONCLUSIONS: Hif-1α and SOX9 proteins can be used as a marker to show severity of preeclampsia and regulation of cell proliferation and angiogenesis during placental development.

Decreased Levels of Soluble Developmental Endothelial Locus-1 Are Associated with Thrombotic Microangiopathy in Pregnancy.

HELLP (Hemolysis, Elevated Liver enzymes and Low Platelets) syndrome is a life-threatening complication of pregnancy, which is often secondary to preeclampsia. To date, there is no biomarker in clinical use for the early stratification of women with preeclampsia who are under increased risk of HELLP syndrome. Herein, we show that the levels of circulating developmental endothelial locus-1 (DEL-1), which is an extracellular immunomodulatory protein, are decreased in patients with HELLP syndrome compared to preeclampsia. DEL-1 levels are also negatively correlated with the circulating levels of kidney injury molecule-1 (KIM-1), which is a biomarker for disorders associated with kidney damage. Receiver-operating characteristic curve analysis for DEL-1 levels and the DEL-1 to KIM-1 ratio demonstrates that these values could be used as a potential biomarker that distinguishes patients with HELLP syndrome and preeclampsia. Finally, we show that placental endothelial cells are a source for DEL-1, and that the expression of this protein in placenta from patients with HELLP syndrome is minimal. Taken together, this study shows that DEL-1 is downregulated in HELLP syndrome both in the circulation and at the affected placental tissue, suggesting a potential role for this protein as a biomarker, which must be further evaluated.

Role of cited-1 and caspase-6 expression in HELLP syndrome.

OBJECTIVE: In this study, we investigated the immunohistochemical staining of cited-1 and caspase-6 expression in the placentas of pregnant women with HELLP syndrome. PATIENTS AND METHODS: Placentas of 20 normotensive patients and 20 women with HELLP syndrome were processed for routine histological tissue processing. The biochemical and clinical parameters of patients were recorded. Placentas were stained with hematoxylin-eosin and cited-1 and caspase-6 immunostaining. RESULTS: Placentas of normotensive patients showed normal histology. Placentas of women with HELLP syndrome showed degenerated cells, hyalinization and vacuolization. Cited-1 expression was negative in normotensive group; however, it was increased in HELLP group, especially in decidual cells, endothelial cells and other placental cells. Caspase-6 expression was negative in placental structures of normotensive groups. However, it was intense in decidual cells, vacuolar and hyalinized areas, inflammatory cells and connective tissue cells in HELLP group. CONCLUSIONS: Cited-1 and caspase-6 are a marker in determining the severity of HELLP syndrome.

Angiotensin-1 and vimentin expression and ultrastructural examination in severe preeclampsia complicated by HELLP syndrome changes in the structure of the umbilical cord.

OBJECTIVE: The purpose of this study was to examine the histopathologic, ultrastructural and immunohistochemical changes in the umbilical cord in women diagnosed with HELLP syndrome. MATERIALS AND METHODS: Postpartum umbilical cords of 40 patients at the 35-38th week of pregnancy were included. 20 severe preeclamptic (HELLP) and 20 normal umbilical cords were used. After the follow-up of tissue parts of 10% formaldehyde solution for histopathology and immunohistochemistry, histopathological and angiopoietin-1 and vimentin antibodies were examined as immunohistochemical after routine paraffin follow-up. For electron microscope analysis, umbilical cord samples were taken into 2.5% glutaral aldehyde solution. RESULTS: In the statistical comparison, mean difference in increased diameter and additional anomaly on the ultrasound of preeclamptic patients was statistically different compared to control patients. In the HELLP group, hyperplasia and degenerative changes, pyknosis of the endothelial cell nuclei of the vessels and apoptotic changes in some regions were observed. Immunohistochemical analysis showed that endothelial cells, basal membrane and fibroblast cells in the HELLP group expressed high levels of vimentin. Angiotensin-1 expression was increased in amniotic epithelial cells, endothelial cells and some pericyte cells. CONCLUSIONS: As a result, it was observed that the signaling that started with trophoblastic invasion with the effect of hypoxia in severe preeclampsia and continued with dysfunction in endothelial cells was parallel to the increase in angiotensin and vimentin receptors. It is thought that the ultrastructural change in endothelial cells may cause disruption of the collagenized structure in Wharton gel, which supports this, and may cause adverse effects in fetal development and nutrition.

Investigation of FAS and IL-6 expression in placentas with HELLP syndrome / Investigación de la expresión de FAS e IL-6 en placentas con síndrome de HELLP

SUMMARY: The aim of our study was to investigate the effect of inflammation in the placenta on the pro-apoptotic development after severe preeclampsia. Placenta tissue samples of 15 HELLP syndrome and 15 healthy 35-38th week-pregnant women were involved in the study. Tissue samples were taken only from the maternal side of the placenta and fixed in 10 % formaldehyde, then blocked in paraffin wax and 4-6 mm-thick sections were cut and stained with Harris Hematoxylene-Eosin. Antigen retrieval was performed for sections, incubated with FAS antibody and anti-IL-6 antibody. After the application of streptavidin peroxidase followed by AEC chromogen solution, sections were counterstained with Harris hematoxylin. Significant thickening of the fibrinoid layer, degeneration and apoptotic change in decidua cells, marked increase in the hyalinized area, degenerative changes in the syncytial regions of the chorionic villus and an increase in syncytial nodes and bridges and IL- expression were observed as positive. FAS expression was positive in the pycnotic nuclei of decidual cells in the maternal region and in the syncytial regions. It was observed that the proapoptotic process increased as a result of severe preeclampsia. It was concluded that the control of cytokine activity and reduction of pro-apoptotic signal during the inflammation process will slow down the development of HELLP syndrome.

RESUMEN: El objetivo de nuestro estudio fue investigar el efecto de la inflamación en la placenta sobre el desarrollo proapoptótico después de la preeclampsia severa. Se recogieron muestras de tejido de placenta de 15 mujeres con síndrome de HELLP y 15 mujeres sanas con un embarazo de 35 a 38 semanas. Se tomaron muestras de tejido solo del lado materno de la placenta y se fijaron en formaldehído al 10 %, luego se bloquearon en parafina y se cortaron secciones de 4-6 mm de espesor y se tiñeron con hematoxilena-eosina de Harris. La recuperación del antígeno se realizó para secciones, incubadas con anticuerpo FAS y anticuerpo anti-IL-6. Después de la aplicación de estreptavidina peroxidasa seguida de solución de cromógeno AEC, las secciones se contrastaron con hematoxilina de Harris. Se observó como positivo un engrosamiento significativo de la capa fibrinoide, degeneración y cambio apoptótico en las células de la decidua, aumento marcado en el área hialinizada, cambios degenerativos en las regiones sincitiales de la vellosidad coriónica y un aumento en los nódulos y puentes sincitiales y la expresión de IL-6. La expresión de FAS fue positiva en los núcleos picnóticos de las células deciduales en la región materna y en las regiones sincitiales. Se observó que el proceso proapoptótico se incrementó como consecuencia de la preeclampsia severa. Se concluyó que el control de la actividad de las citocinas y la reducción de la señal proapoptótica durante el proceso de inflamación ralentizarán el desarrollo del síndrome de HELLP.

Abnormal expression of the costimulatory molecule B7-H4 in placental chorionic villous and decidual basalis tissues of patients with preeclampsia and HELLP syndrome.

BACKGROUND: B7-H4, a checkpoint molecule of the B7 family, regulates a broad spectrum such as T-cell activation, cytokine secretion, tumour progression, and invasion capacities. Our previous data revealed that soluble B7-H4 (sB7-H4) blood serum levels are elevated in women at high risk for the hypertensive pregnancy disorder preeclampsia (PE) in the first trimester, as well as in patients with confirmed early/late-onset PE. AIM: We here aim to investigate the expression pattern of B7-H4 in placental tissues of PE and HELLP Syndrome versus control group. METHODS: B7-H4 protein expression and localization were investigated by immunoblotting and co-immunohistochemistry in placental chorionic villous and decidual basalis tissues. RESULTS: B7-H4 protein was prominently expressed at the cell membrane, in the cytoplasm of the syncytiotrophoblast (STB) and interstitial extravillous trophoblast (EVT). B7-H4 protein levels in placental chorionic villous tissue were significantly higher in women with early-onset/late-onset PE and HELLP, while it was decreased in decidual basalis tissues of early-onset PE and HELLP compared with controls. CONCLUSION: B7-H4 was inversely expressed in placental chorionic villous and decidual basalis tissues of PE and HELLP patients. The increase in B7-H4 in the STB in PE and HELLP may lead to excessive apical expression and release of soluble B7-H4 in the maternal circulation. In contrast, the decrease in B7-H4 in decidual basalis tissues could be related to the decrease in invasion ability of the EVT in PE. Thus, the current results strongly suggest that B7-H4 is involved in the pathogenesis of PE and HELLP.

Transcriptome analysis of placentae reveals HELLP syndrome exhibits a greater extent of placental metabolic dysfunction than preeclampsia.

Objective: The association between HELLP syndrome and preeclampsia has been investigated with conflicting conclusions. This study is to investigate the pathogenesis underlying these two diseases by analyzing placental transcriptome.Methods: The gene expression profile downloaded from Gene Expression Omnibus database was analyzed by R language.Results: A total of 573 differentially expressed genes in HELLP syndrome and 358 in preeclampsia were identified, among which 295 were unique to HELLP syndrome. Some metabolism-associated pathways were uniquely enriched in HELLP syndrome.Conclusions: HELLP syndrome exhibits a greater extent of placental metabolic dysfunction than preeclampsia, although these two diseases might share partial pathogenesis.

Inhibiting PDGF-D alleviates the symptoms of HELLP by suppressing NF-κB activation.

Hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome is a life-threatening pregnancy complication. Though there are several medications widely used to treat HELLP syndrome, delivery is the only efficient treatment. The goal of the present study was to investigate the effects of platelet-derived growth factor-D (PDGF-D), a newly identified PDGF, in a rat model of HELLP syndrome which was accomplished by sFlt-1 and sEng injection. The expression levels of PDGF-D in pregnant women diagnosed with HELLP syndrome was determined. A HELLP rat model was established and the PDGF-D expression level in the plasma and the placenta tissue was evaluated. To evaluate the effects of PDGF-D in HELLP syndrome model, siPDGF-D was injected into the rats and the HELLP syndrome-related parameters were measured. The levels of inflammatory cytokines and PDGF-D were determined by ELISA. The oxidative stress activities in the plasma were also determined. Furthermore, the expression of PDGF-D/PDGFR-ß/nuclear factor κB (NF-κB) p65 in placenta tissues was evaluated by Western blotting. Compared to the normal pregnant (NP) group, the levels of PDGF-D were augmented regardless of species. Knockdown of PDGF-D can result in the alleviation of HELLP syndrome development and progression in the HELLP rat model. Importantly, as a result of PDGF-D knockdown, the serum levels of inflammatory cytokines and oxidative stress activities were modulated, and the phosphorylation of PDGFR-ß and NF-κB p65 in placenta tissue was inhibited. Taking together, our findings indicate that targeting PDGF-D could be used as a novel strategy to treat patients with HELLP syndrome.

Role of placental inflammatory mediators and growth factors in patients with rheumatic diseases with a focus on systemic sclerosis.

OBJECTIVES: Pregnancy in SSc is burdened with an increased risk of obstetric complications. Little is known about the underlying placental alterations. This study aimed to better understand pathological changes and the role of inflammation in SSc placentas. Leucocyte infiltration, inflammatory mediators and atypical chemokine receptor 2 (ACKR2) expression in SSc placentas were compared with those in other rheumatic diseases (ORD) and healthy controls (HC). METHODS: A case-control study was conducted on eight pregnant SSc patients compared with 16 patients with ORD and 16 HC matched for gestational age. Clinical data were collected. Placentas were obtained for histopathological analysis and immunohistochemistry (CD3, CD20, CD11c, CD68, ACKR2). Samples from four SSc, eight ORD and eight HC were analysed by qPCR for ACKR2 expression and by multiplex assay for cytokines, chemokines and growth factors involved in angiogenesis and inflammation. RESULTS: The number of placental CD3, CD68 and CD11 cells was significantly higher in patients affected by rheumatic diseases (SSc+ORD) compared with HC. Hepatocyte growth factor was significantly increased in the group of rheumatic diseases patients (SSc+ORD) compared with HC, while chemokine (C-C motif) ligand 5 (CCL5) was significantly higher in SSc patients compared with ORD and HC. CCL5 levels directly correlated with the number of all local inflammatory cells and higher levels were associated with histological villitis. CONCLUSIONS: Inflammatory alterations characterize placentas from rheumatic disease patients and could predispose to obstetric complications in these subjects.

The clinical course of COVID in pregnancy.

The 2019 novel coronavirus disease (COVID-19) pandemic poses unique challenges to the medical community as the optimal treatment has not been determined and is often at the discretion of institutional guidelines. Pregnancy has previously been described as a high-risk state in the context of infectious diseases, given a particular susceptibility to pathogens and adverse outcomes. Although ongoing studies have provided insight on the course of this disease in the adult population, the implications of COVID-19 on pregnancy remains an understudied area. The objective of this study is to review the literature and describe clinical presentations among pregnant women afflicted with COVID-19.

Pre-eclampsia-like syndrome induced by severe COVID-19: a prospective observational study.

OBJECTIVES: To investigate the incidence of clinical, ultrasonographic and biochemical findings related to pre-eclampsia (PE) in pregnancies with COVID-19, and to assess their accuracy to differentiate between PE and the PE-like features associated with COVID-19. DESIGN: A prospective, observational study. SETTING: Tertiary referral hospital. PARTICIPANTS: Singleton pregnancies with COVID-19 at >20+0  weeks. METHODS: Forty-two consecutive pregnancies were recruited and classified into two groups: severe and non-severe COVID-19, according to the occurrence of severe pneumonia. Uterine artery pulsatility index (UtAPI) and angiogenic factors (soluble fms-like tyrosine kinase-1/placental growth factor [sFlt-1/PlGF]) were assessed in women with suspected PE. MAIN OUTCOME MEASURES: Incidence of signs and symptoms related to PE, such as hypertension, proteinuria, thrombocytopenia, elevated liver enzymes, abnormal UtAPI and increased sFlt-1/PlGF. RESULTS: Thirty-four cases were classified as non-severe and 8 as severe COVID-19. Five (11.9%) women presented signs and symptoms of PE, all five being among the severe COVID-19 cases (62.5%). However, abnormal sFlt-1/PlGF and UtAPI could only be demonstrated in one case. One case remained pregnant after recovery from severe pneumonia and had a spontaneous resolution of the PE-like syndrome. CONCLUSIONS: Pregnant women with severe COVID-19 can develop a PE-like syndrome that might be distinguished from actual PE by sFlt-1/PlGF, LDH and UtAPI assessment. Healthcare providers should be aware of its existence and monitor pregnancies with suspected pre-eclampsia with caution. TWEETABLE ABSTRACT: This study shows that a pre-eclampsia-like syndrome could be present in some pregnancies with severe COVID-19.

Effects of preeclampsia and eclampsia on maternal metabolic and biochemical outcomes in later life: a systematic review and meta-analysis.

OBJECTIVE: To evaluate the association between preeclampsia (PE) and eclampsia (E) on subsequent metabolic and biochemical outcomes. METHODS: Systematic review and meta-analysis of observational studies. We searched five engines until November 2018 for studies evaluating the effects of PE/E on metabolic and biochemical outcomes after delivery. PE was defined as presence of hypertension and proteinuria at >20 weeks of pregnancy; controls did not have PE/E. Primary outcomes were blood pressure (BP), body mass index (BMI), metabolic syndrome (MetS), blood lipids and glucose levels. Random effects models were used for meta-analyses, and effects reported as risk difference (RD) or mean difference (MD) and their 95% confidence interval (CI). Subgroup analyses by time of follow up, publication year, and confounder adjustment were performed. RESULTS: We evaluated 41 cohorts including 3300 PE/E and 13,967 normotensive controls. Women were followed up from 3 months after delivery up to 32 years postpartum. In comparison to controls, PE/E significantly increased systolic BP (MD = 8.3 mmHg, 95%CI 6.8 to 9.7), diastolic BP (MD = 6.8 mmHg, 95%CI 5.6 to 8.0), BMI (MD = 2.0 kg/m2; 95%CI 1.6 to 2.4), waist (MD = 4.3 cm, 95%CI 3.1 to 5.5), waist-to-hip ratio (MD = 0.02, 95%CI 0.01 to 0.03), weight (MD = 5.1 kg, 95%CI 2.2 to 7.9), total cholesterol (MD = 4.6 mg/dL, CI 1.5 to 7.7), LDL (MD = 4.6 mg/dL; 95%CI 0.2 to 8.9), triglycerides (MD = 7.7 mg/dL, 95%CI 3.6 to 11.7), glucose (MD = 2.6 mg/dL, 95%CI 1.2 to 4.0), insulin (MD = 19.1 pmol/L, 95%CI 11.9 to 26.2), HOMA-IR index (MD = 0.7, 95%CI 0.2 to 1.2), C reactive protein (MD = 0.05 mg/dL, 95%CI 0.01 to 0.09), and the risks of hypertension (RD = 0.24, 95%CI 0.15 to 0.33) and MetS (RD = 0.11, 95%CI 0.08 to 0.15). Also, PE/E reduced HDL levels (MD = -2.15 mg/dL, 95%CI -3.46 to -0.85). Heterogeneity of effects was high for most outcomes. Risk of bias was moderate across studies. Subgroup analyses showed similar effects as main analyses. CONCLUSION: Women who had PE/E have worse metabolic and biochemical profile than those without PE/E in an intermediate to long term follow up period.

Placental fibronectin staining is unaffected in pregnancies complicated by late-onset intrauterine growth restriction and small for gestational age fetuses.

OBJECTIVE: To investigate the placental staining pattern of fibronectin, an extracellular matrix protein essential for trophoblastic invasion, in pre-eclampsia and fetal growth restriction. METHODS: This was a retrospective study conducted at the University of Udine, including the placentas of women with pre-eclampsia and fetal growth restriction collected between January 1, 2001, and December 31, 2010. Fibronectin was evaluated in placental tissue micro-array by immunohistochemistry, describing localization and intensity of staining. RESULTS: The study included the placentas of 36 women with early-onset (delivery <34 weeks of gestation) pre-eclampsia; 6 with early-onset HELLP syndrome; 17 with early-onset intrauterine growth restriction (IUGR); 14 with late-onset (delivery ≥34 weeks of gestation) pre-eclampsia; 35 with late-onset IUGR; 18 with small for gestational age (SGA) fetuses (birth weight <10th percentile); and 64 controls. Fibronectin was present both at the cell surface and in the cytoplasm. Cytoplasm staining intensity resulted higher in early forms of pregnancy-related complications compared to controls, although this was statistically significant (P<0.05) only for early-onset pre-eclampsia (P=0.085 for HELLP syndrome; P=0.091 for IUGR). Also, late-onset forms of pre-eclampsia had stronger cytoplasmic and pericellular staining compared to controls (P<0.05). Interestingly, staining of both late-onset IUGR and SGA was comparable to controls. CONCLUSION: Fibronectin appeared to be unaffected in women with late-onset IUGR and SGA fetuses, suggesting a peculiar common pathogenetic pattern in these conditions.

Angiogenic profiling in HELLP syndrome cases with or without hypertension and proteinuria.

OBJECTIVES: Angiogenic profiling with the use of sFlt-1/PlGF ratio (soluble fms-like tyrosine kinase-1/placental growth factor) can be helpful to characterize women with signs of impending preeclampsia (PE). However, little is known about the angiogenic profile of pregnancies complicated by HELLP syndrome. The aim of this study was to examine the relationship of angiogenic profiles in cases of HELLP syndrome with and without classical signs of preeclampsia. STUDY DESIGN: The angiogenic profile of pregnant women with singleton gestation and isolated PE (group 1), PE associated with HELLP syndrome (group 2), and isolated HELLP syndrome (group 3) from 01/2011 to 03/2018, were compared. To overcome gestational age dependent angiogenic behavior, cases (group 3) were matched 1:2 with cases from group 1 and 2. Matching criteria was gestational age (±1 week). PE and HELLP syndrome were defined according to the international Society for the Study of Hypertension in Pregnancy (ISSHP) statement 2014. RESULTS: During the observational period, 244 women could be included in the study. Of those, 237 (97.1%) were diagnosed with PE. In 42 cases (17.2%) PE was associated with HELLP syndrome while 7 (2.9%) patients were diagnosed with isolated HELLP syndrome. Angiogenic profiles in terms of sFlt-1/PlGF ratios differed significantly between the three groups, showing highest levels in group 2 (PE/HELLP) while cases with isolated HELLP demonstrated the lowest ratios and sFlt-1 values (p = 0.01). CONCLUSION: We conclude that isolated HELLP syndrome is rare and seems to be a particular entity expressing a different angiogenic behaviour compared to classical PE or PE associated with HELLP syndrome.

Placental sFLT1 is associated with complement activation and syncytiotrophoblast damage in preeclampsia.

The immune complement system protects against pathogens; however, excess activation results in disease like hemolytic uremic syndrome, a clinical imitator of preeclampsia. Vascular endothelial factor (VEGF) protects against aberrant complement activation and is inhibited by soluble fms-like tyrosine kinase-1 (sFLT1) in other organs. We hypothesize that sFLT1 promotes complement-mediated placental damage through VEGF inhibition in preeclampsia. Objective: Quantify placental complement activity and sFLT1 expression in preeclampsia, and the subgroup of preeclampsia with hemolysis elevated liver enzymes low platelets (HELLP) syndrome. Methods: Placental complement activation marker C4d, membrane attack complex (MAC), and sFLT1 expression was quantified using immunofluores cence microscopy. Results: Placentas from 18 controls, 25 preeclampsia, including 6 cases of HELLP syndrome were identified. Placental C4d expression was greater in PE (median 6.4 [IQR: 5.1, 8.3]) compared to controls (4.4 [3.6, 5.5]; p = 0.003). MAC expression was also increased in preeclampsia compared to controls (6.5 [5.8, 8.7]; 5.4 [2.9, 5.9], p = 0.001). Placental sFLT1 expression was also higher in preeclampsia (p <0.0001). C4d and MAC were strongly correlated with sFLT1 levels in the placenta (R = 0.72; p < 0.0001 and R = 0.59; p = 0.01, respectively). Complement and sFLT1 expression was elevated in HELLP compared to preeclampsia without laboratory abnormalities, but this difference did not reach statistical significance. Conclusion: Increased placental complement activation and damage was seen in preeclampsia and correlates with sFLT1 expression. Our findings support the importance of the complement pathway in preeclampsia.

Recurrent Placental Transcriptional Profile With a Different Histological and Clinical Presentation: A Case Report.

Statistically, patients with severe pregnancy complications are at risk of recurrent complications, but it is less understood if patients present with similar or different placental pathologies in subsequent pregnancies. In this case report, we describe 2 consecutive adverse pregnancies in the same woman 4 years apart. The first pregnancy was diagnosed as early-onset preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome, with placental maternal vascular malperfusion features, such as syncytial knots and accelerated villous maturity. In contrast, the second pregnancy was associated with normotensive fetal growth restriction and placental "immunological" lesions, such as massive perivillous fibrin deposition and chronic intervillositis. However, based on the expression of FLT1, LIMCH1, and TAP1 by quantitative polymerase chain reaction, the placentas from both pregnancies were found to exhibit an "immunological" transcriptional signature. This suggests that this small panel of gene expression markers may be able to predict the future reoccurrence of an immunological placental pathology despite no histological evidence within the first pregnancy. These results call for more studies looking at paired pregnancies of individuals with recurrent obstetric complications and confirm the importance of assessing matched transcriptional and histopathological placental information.

Increased placental expression of Placental Protein 5 (PP5) / Tissue Factor Pathway Inhibitor-2 (TFPI-2) in women with preeclampsia and HELLP syndrome: Relevance to impaired trophoblast invasion?

INTRODUCTION: Placental Protein 5 (PP5)/Tissue Factor Pathway Inhibitor-2 (TFPI-2) is an extracellular matrix-associated protein mainly expressed by the syncytiotrophoblast that may regulate trophoblast invasion. Our aim was to study placental PP5/TFPI-2 expression and its relation to placental pathology in various forms of preeclampsia and HELLP syndrome. METHODS: Placental and maternal blood specimens were collected at the time of delivery from the same women in the following groups: 1) early controls; 2) early preeclampsia; 3) early preeclampsia with HELLP syndrome; 4) late controls; and 5) late preeclampsia. After histopathological examination, placental specimens were immunostained with polyclonal anti-PP5/TFPI-2 antibody on Western blot and tissue microarray immunohistochemistry. Placental PP5/TFPI-2 immunoscores were assessed manually and with a semi-automated method. Maternal sera were immunoassayed for PP5/TFPI-2. RESULTS: PP5/TFPI-2 was localized to the cytoplasm of syncytiotrophoblast. Manual and semi-automated PP5/TFPI-2 immunoscores were higher in early preeclampsia with or without HELLP syndrome but not in late preeclampsia than in respective controls. In patients with preeclampsia, the correlation of placental PP5/TFPI-2 expression with maternal vascular malperfusion score of the placenta was positive while it was negative with birthweight and placental weight. Maternal serum PP5/TFPI-2 concentration was higher in early preeclampsia and it tended to be higher in early preeclampsia with HELLP syndrome than in early controls. DISCUSSION: Our findings suggest that an increased placental PP5/TFPI-2 expression may be associated with abnormal placentation in early preeclampsia, with or without HELLP syndrome.

Development and validation of GC-MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: evidence of altered methylation, transamidination, and arginase activity.

We developed and validated gas chromatography-mass spectrometry (GC-MS) methods for the simultaneous measurement of amino acids and their metabolites in 10-µL aliquots of human plasma and urine. De novo synthesized trideutero-methyl esters were used as internal standards. Plasma proteins were precipitated by acidified methanol and removed by centrifugation. Supernatants and native urine were evaporated to dryness. Amino acids were first esterified using 2 M HCl in methanol and then amidated using pentafluoropropionic anhydride for electron-capture negative-ion chemical ionization. Time programmes were used for the gas chromatograph oven and the selected-ion monitoring of specific anions. The GC-MS methods were applied in clinical studies on the HELLP syndrome and pediatric kidney transplantation (KTx) focusing on L-arginine-related pathways. We found lower sarcosine (N-methylglycine) and higher asymmetric dimethylarginine (ADMA) plasma concentrations in HELLP syndrome women (n = 7) compared to healthy pregnant women (n = 5) indicating altered methylation. In plasma of pediatric KTx patients, lower guanidinoacetate and homoarginine concentrations were found in plasma but not in urine samples of patients treated with standard mycophenolate mofetil-based immunosuppression (MMF; n = 22) in comparison to matched patients treated with MMF-free immunosuppression (n = 22). On average, the global arginine bioavailability ratio was by about 40% lower in the MMF group compared to the EVR group (P = 0.004). Mycophenolate, the major pharmacologically active metabolite of MMF, is likely to inhibit the arginine:glycine amidinotransferase (AGAT), and to enhance arginase activity in leukocytes and other types of cell of MMF-treated children.

Circulating parameters of oxidative stress and hypoxia in normal pregnancy and HELLP syndrome.

BACKGROUND: The HELLP syndrome (Hemolysis, Elevated Liver enzymes and Low Platelets) is a complication of severe pre-eclampsia, a condition characterized by oxidative stress elevation caused by disequilibrium between lipid peroxidation and antioxidant defense mechanisms, which, in turn, results in endothelial compromise and free radical-mediated cell damage. While several studies have examined the relationship between pre-eclampsia and oxidative stress, research investigating oxidative and hypoxic status in HELLP syndrome is limited. OBJECTIVES: The aim of this study was to compare the levels of oxidative stress markers - total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), and malondialdehyde (MDA) - and a hypoxia marker - carbonic anhydrase IX (CA IX) - in patients with HELLP syndrome and in healthy pregnant women. MATERIAL AND METHODS: A total of 23 women with HELLP syndrome and 30 healthy pregnant women were included in the study. Serum levels of oxidative stress markers were determined using colorimetric methods, while serum levels of CA IX were measured using an enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: The TOS, OSI, MDA, and CA IX levels were significantly higher in women with HELLP syndrome than in the controls (p = 0.0001, p = 0.0001, p = 0.0001, and p = 0.008, respectively). CONCLUSIONS: Increased levels of oxidative stress and hypoxia markers in women with HELLP syndrome suggest that oxidative stress and hypoxia may be significantly involved in the pathophysiology of the disease. Further follow-up studies are now needed to investigate the prognostic roles of these parameters in patients with HELLP syndrome.

Attenuation of oxidative stress and hypertension in an animal model of HELLP syndrome.

HELLP (hemolysis elevated liver enzyme low platelet) syndrome is associated with hypertension, inflammation, oxidative stress and endothelial activation. The objective of this study was to determine if oxygen scavenging or endothelin A receptor antagonism improved hypertension and oxidative stress. sFlt-1 and sEndoglin were infused via mini-osmotic pump into normal pregnant rats (NP) on gestational day 12 to create HELLP syndrome. On gestational day 18 arterial catheters were inserted and on gestational day 19 mean arterial pressure was analyzed in rats; serum, urine and tissues were collected for molecular analysis. HELLP rats had significantly increased MAP compared to control normal pregnant rats (P < 0.0005). Endothelin A receptor antagonism via ABT-627 and Tempol, superoxide dismutase mimetic, were administered to a subset of normal pregnant and HELLP rats beginning on gestational day 13 and attenuated mean arterial pressure in HELLP rats (P < 0.05; P < 0.005). There were no statistically significant differences in mean arterial pressure between NP+ETA Receptor or NP+Tempol treated rats and NP rats (P = 0.22). Endothelin A receptor blockade significantly decreased HELLP induced isoprostane excretion (P < 0.0005), placental and hepatic reactive oxygen species (P < 0.05; P < 0.0005) and increased placental total antioxidant capacity (P < 0.005) compared to untreated HELLP rats. Similar results in isoprostane (P < 0.005), hepatic reactive oxygen species (P < 0.05) and placental total antioxidant capacity (P < 0.05) were seen in HELLP rats treated with Tempol or Endothelin A receptor antagonist vs. untreated HELLP rats. These data demonstrated a role for oxidative stress in contributing to the hypertension, placental and liver damage that is seen in HELLP syndrome.