Nucleic acids and protein changes in normal and pre-eclamptic placentae.

DNA and RNA assayed in the placentae of three groups of pregnant women: normal second trimester (16-28), normal third trimester (28 weeks up to term) and in preeclampsia. The protein level in the placentae of the three groups was also assayed. The proteins, DNA and RNA all decreased after 28 weeks and up to term in normal pregnancy. In pre-eclampsia DNA and RNA showed a significant increase compared with cases of normal third trimester pregnancy. Whereas the proteins also showed an increased level, this was still less than its concentration during the second trimester of pregnancy. Protein/DNA and RNA/DNA ratios were calculated for the three groups. These ratios showed a gradual decrease during normal pregnancy from 16 weeks up to term, but with a sharper decrease in pre-eclampsia.     

Dynamic thiol/disulphide homeostasis and ischemic modified albumin levels in isolated oligohydramnios

ObjectiveOligohydramnios is defined as amniotic fluid index in ultrasonographic measurement is less than 5 percentile according to gestational age, the amniotic fluid volume is ≤ 5 cm, or if the single deepest dial is < 2 cm. The condition of oligohydramnios that not with fetal structural/chromosomal abnormalities, intrauterine growth retardation, intrauterine infection and maternal disease is described as isolated oligohydramnios (IO). The aim of this study is to examine whether oxidative stress and reactive oxygen species (ROS) have a place in the pathophysiology of IO.Materials and methodsIn this prospective case–control study, a total of 126 participants were included. The patient group consisted of 65 patients who were diagnosed IO, and the control group consisted of 61 healthy normal pregnants. Native thiol (-SH), total thiol (-SH + -SS), dynamic disulfide (-SS), IMA values from maternal serum were measured and compared between groups.ResultsMaternal serum -SH and -SH + -SS values were significantly lower in the IO group than in the control group (409.47 ± 55.36 μmol/L vs. 437.40 ± 48.68 μmol/L, p = 0.03 and 457.40 ± 63.01 μmol/L vs. 484.59 ± 52.75 μmol/L, p = 0.01). In the IO group when -SS/-SH and -SS/-SH + -SS ratio was found to be statistically significantly higher than control group (5.84 ± 1.1 vs 5.41 ± 0.71, p = 0.01 and 5.2 ± 0.88 vs 4.8 ± 0.58, p = 0.01), -SH/-SH + -SS ratio was significantly lower (89.56 ± 1.7 vs 90.24 ± 1.16, p = 0.01). There was no significant difference in terms of -SS value (p = 0.66). IMA value was significantly higher in the IO group than control group (0.76 ± 0.10 ABSU vs 0.68 ± 0.06, p < 0.01). It is seen as a result of ROC analysis that -SH, -SH + -SS, -SS/-SH, -SS/-SH + -SS, -SH/-SH + -SS and IMA values have a diagnostic value for IO (p < 0.05).ConclusionThe thiol/disulfide balance shifted towards oxidative stress in IO compared to control group. So oxidative stress and ROS have a place in the pathophysiology of IO.     

Immunohistochemical localization of pregnancy-associated plasma protein A (PAPP-A) in placentae from normal and pre-eclamptic pregnancies

An immunohistochemical method was used to locate pregnancy-associated plasma protein A (PAPP-A) in the placenta and uterus. In addition to 10 placentae and basal plates from normal pregnancies, ranging in gestational age from 37 to 40 weeks, the following specimens were studied: three uteri obtained by hysterectomy during early pregnancy; and three placentae from patients with severe hypertensive pre-eclampsia. In early gestation, PAPP-A was localized in the villous cytotrophoblastic cell layer and the endometrial glands but was not found in the villous syncytiotrophoblast, the cytotrophoblastic cell columns or the decidual cells. On histochemical examination of placentae from cases of pre-eclampsia with hypertension, an increased number of villous cytotrophoblastic cells and so-called X-cells was observed. The monospecific antiserum to PAPP-A reacted strongly and evenly with the cytoplasm of these cells. The present study strongly suggests that the active production sites of PAPP-A are the villous cytotrophoblastic cells and the X-cells.     

Monoamine oxidase expression and activity in human placentae from pre-eclamptic and normotensive pregnancies

A feature of pre-eclampsia is that circulating levels of maternal serotonin (5-hydroxytryptamine) are elevated and placental monoamine oxidase-A (MAO-A) activity, the major factor in the regulation of serotonin levels in pregnancy, is reduced. It is not known whether this is due to a reduced MAO-A protein content or a reduced catalytic turnover of the serotonin by MAO-A; this question has been addressed in the present work. Term placentae from normotensive and pre-eclamptic women were analysed for MAO-A specific mRNA expression (by semi-quantitative RT-PCR), MAO-A protein (by immunohistochemistry and quantitative ELISA, using a MAO-A specific monoclonal antibody), together with MAO activity (using [(3)H] labelled 5-hydroxytryptamine as substrate). Immunohistochemical analysis of placentae from both normotensive and pre-eclamptic women demonstrated that MAO-A protein is located in the cytoplasm of the placental syncytiotrophoblast layer, consistent with a mitochondrial location; no MAO-A protein was found in the nucleus. No MAO-B protein was detected in this placental layer, despite the presence of MAO-B mRNA. The results indicate that both total protein/g fresh weight and MAO-A protein/g fresh weight were approximately 40 per cent lower in pre-eclamptic than in normotensive placentae, but that there was no statistical difference in the expression of MAO-A mRNA in relation to GAPDH or actin mRNA or in MAO-A protein/mg total protein. However, MAO-A activity/g fresh weight was significantly reduced in pre-eclamptic placentae, in agreement with previous findings. This was found to be due to a 60 per cent reduction (P< 0.05) in the catalytic turnover (activity/molecule) of the enzyme. This study has therefore clearly shown that the expression of placental MAO-A specific mRNA and MAO-A protein are not specifically affected in pre-eclampsia, but that the catalytic efficiency of the expressed MAO-A enzyme in pre-eclamptic placentae is greatly reduced.     

Investigation of serum thiol/disulphide homeostasis in patients with abortus imminens

Aim: The aim of our study is to compare serum thiol/disulphide homeostasis of women diagnosed with abortus imminens (AI) and healthy pregnant women, and to determine whether it has a role in the pathogenesis of the disease or not.

Materials and methods: A total of 100 pregnant women were included in the study. The study group consisted of 50 patients with AI whereas 50 healthy pregnant women were chosen as the control group. All of the patients in the two groups were matched for age, gestational age, and body mass index. Thiol/disulphide levels were analyzed with a newly developed automated spectrophotometric method.

Results: We found significantly reduced levels of native thiol (SH) (370.2?±?35.2?μmol/l versus 397.6?±?29.3?μmol/l) and total thiol (406?±?37.1?μmol/l versus 434.5?±?29.9?μmol/l), in the sera of the study group compared to the control group (p?p: .612). Similarly, no significant differences were observed between the two groups in terms of SS/SH, SS/total thiol and SH/total thiol ratios (all p?>?.05).

Conclusions: We found a decrease in thiol levels which have antioxidant properties in patients with AI. Decreased antioxidant level in the body is thought to play a role in the etiology of AI. In this regard, further studies are needed to elucidate the potential role of dynamic thiol/disulphide homeostasis in the pathogenesis of AI.     

Enhancement of mitochondrial oxidative stress and up-regulation of antioxidant protein peroxiredoxin III/SP-22 in the mitochondria of human pre-eclamptic placentae

A growing body of evidence indicates that the pathogenesis of pre-eclampsia is closely associated with oxidative stress occurring in mitochondria. In the present study, we evaluated the degree of mitochondrial lipid peroxidation by assessing the accumulation of 4-hydroxy-2-nonenal (HNE)-modified proteins and examined the expression of mitochondrial antioxidant protein peroxiredoxin III/SP-22 in normal and pre-eclamptic human placentae. The accumulation of HNE-modified proteins increased to a greater extent in both the mitochondria and cytosol of pre-eclamptic placentae than in those of normal placentae. Moreover, the accumulation of HNE-modified proteins was much more evident in the mitochondria than in the cytosol, indicating that lipid peroxidation occurred mainly in the mitochondria of pre-eclamptic placentae. The mRNA expression of peroxiredoxin III/SP-22 was increased about 2-fold in pre-eclamptic placentae compared to normal placentae. The protein levels of peroxiredoxin III/SP-22 were approximately 4-fold higher in pre-eclamptic placentae than in normal placentae. Immunohistochemistry of placental tissues showed that the levels of peroxiredoxin III/SP-22 protein were increased in the trophoblasts of floating villi, stromal cells of stem villi, and decidual cells in pre-eclamptic placentae. These results indicate that peroxiredoxin III/SP-22 plays a crucial role in the protection of placental function from oxidative stress occurring in mitochondria of pre-eclamptic placentae.     

Reducing agent and tunicamycin-responsive protein (RTP) mRNA expression in the placentae of normal and pre-eclamptic women

Recently, the gene encoding a new stress-induced protein termed reducing agent and tunicamycin-responsive protein (RTP) was identified. The function of RTP is unknown, however, the strong upregulation of RTP during cellular differentiation, and exposure to stress conditions including hypoxia suggests a specific role for RTP in these processes. In pre-eclampsia, impaired spiral artery remodelling and reduced perfusion may reduce oxygen tension in the placenta and thereby alter trophoblast differentiation and function. We therefore hypothesized that the expression of RTP mRNA is altered in the placentae of women with pre-eclampsia. The aims of this study were to determine the regional distribution and cellular localization of RTP mRNA expression and compare mRNA abundance in different regions of normotensive control and pre-eclamptic placentae. In normal and pre-eclamptic placentae, RTP mRNA was expressed in the syncytiotrophoblasts and in the intermediate trophoblasts of the basal plate. In early onset pre-eclampsia, RTP mRNA was more abundant in the chorionic villi regions. A further increase was localized to the syncytial knots and to the trophoblasts in the peri-infarct regions. The increased RTP expression may reflect lower oxygen tension and/or other stress stimuli in the placenta in pre-eclampsia.     

Regulation of nitric oxide synthase activity by tetrahydrobiopterin in human placentae from normal and pre-eclamptic pregnancies

The possible regulatory role of tetrahydrobiopterin (BH(4)) in Type III nitric oxide synthase (NOS III) activity of human placentae from first trimester, term and pre-eclamptic pregnancies was investigated. In homogenates of first-trimester or term placentae, BH(4)stimulated NOS III activity up to 2.5-fold in a concentration dependent manner from 20 n m to 1 microm BH(4), and half-maximal stimulation (EC(50)) was observed at 100-110 n m. No significant further stimulation was detectable over an extended concentration range from 1 microm to 50 microm BH(4). NOS III present in microsomal and gel-filtered cytosol fractions exhibited similar BH(4)-activation patterns, with an identical EC(50)value of 50 n m. Remarkably, tissue concentrations of BH(4)showed a marked decrease in term placentae (57+/-23 n m, mean+/-s.d., n=26) relative to first-trimester placentae (189+/-79 n m, mean+/-s.d., n=17), suggesting that alterations in cellular BH(4)concentrations may play a more significant role in the regulation of NOS III activity in late pregnancy. Placental homogenates from 10 pre-eclamptic pregnancies exhibited two distinct types of response to BH(4). In seven placental homogenates, addition of physiological concentrations of BH(4)(20 n m to 1 microm) elicited no increase whatsoever in basal NOS III activity, and only high BH(4)concentrations (50 microm) caused notable stimulation (BH(4)resistant group). In contrast, in three of 10 placental homogenates both physiological and 50 microm BH(4)concentrations stimulated NOS III to levels similar to that of normal placentae (BH(4)responsive group). There were no appreciable differences in the clinical presentation of pre-eclampsia between the two groups. Importantly, BH(4)concentrations in pre-eclamptic placentae were comparable with those of normal, control placentae. Taken together, the observations suggest that BH(4)controls NOS III activity in the human placenta, and a defect in BH(4)regulation of NOS III may contribute to the development of pre-eclampsia. A model implicating the malfunction of placental NOS III rather than its actual tissue level in the pathogenesis of pre-eclampsia is discussed.     

Vascular endothelial growth factor expression is unaltered in placentae and myometrial resistance arteries from pre-eclamptic patients

BACKGROUND: The purpose of this study is to determine whether there is any difference for vascular endothelial growth factor expression in placentae and myometrial resistance arteries from control and pre-eclamptic patients to clarify the source of the increased total vascular endothelial growth factor in pre-eclampsia. METHODS: With placentae tissue samples and small pieces of myometrium from the controls (n=10) and pre-eclampsia patients (n=7), vascular endothelial growth factor immunohistochemistry and mRNA in situ hybridization were performed. We also measured the serum vascular endothelial growth factor levels by competitive enzyme immunoassay at the same time. RESULTS: Vascular endothelial growth factor was identified primarily in syncytiotrophoblast in placental tissue, and it was also identified in smooth muscle cells in the myometrium and perivascular smooth muscle in myometrial resistance arteries. However, there were no differences in vascular endothelial growth factor expression between the groups in the presence of elevated serum levels of total vascular endothelial growth factor in pre-eclamptic patients (median 18.2 ng/ml versus 4.9 ng/ml). CONCLUSION: This study suggests that the placenta and perivascular smooth muscle are not the origin of the increased total vascular endothelial growth factor in pre-eclampsia. To clearly understand the role of vascular endothelial growth factor in pre-eclampsia, further studies are required to determine the sites of increased vascular endothelial growth factor synthesis.     

Severely reduced presence of tissue macrophages in the basal plate of pre-eclamptic placentae

Pre-eclampsia is a disorder of unknown aetiology peculiar to human pregnancy. A well-described pathological feature being shallow trophoblast invasion into the spiral arteries during placenta development. Epidemiological studies have revealed an increased risk in pregnancies of primipaternity, and an association with the maternal-fetal HLA-DR relationship, both suggesting the involvement of an immunological component. We were therefore interested in the distribution of HLA-DR expressing myeloid cells in the decidua of healthy and pre-eclamptic placentae. We have studied the monocytes in maternal and fetal peripheral blood as well as in the placenta and identified the cluster of differentiation (CD) 14(+)myeloid cells in the basal plate as mannose receptor (ManR) positive tissue macrophages. In a comparison between peripheral blood monocytes from healthy pregnant and pre-eclamptic women we found no significant difference in the subpopulation size of CD14(+)/CD16(+)monocytes. The number and location of macrophages in the placental villi was similar. However, while the basal plate of the normal decidua contained numerous CD14(+), HLA-DR(bright), ManR(+)tissue macrophages, this compartment was virtually void of these phagocytic cells in the pre-eclamptic placenta. This novel finding suggests that in pre-eclampsia not only the migration of endovascular cytotrophoblasts is disturbed, but that also maternal macrophage migration is affected.     

Localization of indoleamine 2,3-dioxygenase and 4-hydroxynonenal in normal and pre-eclamptic placentae

This study was undertaken to compare placental levels of 2,3-Dioxygenase (IDO), a free radical scavenger, and 4-Hydroxynonenal (4-HNE), a major by-product of lipid peroxidation, in normal and pre-eclamptic pregnancies. Placentae were collected at caesarean section from women with a term, normal singleton pregnancy (37-40 weeks' gestation, n=10) and women with a term singleton pregnancy complicated by pre-eclampsia (n=10). IDO and 4-HNE localization and intensity was studied by semi-quantitative immunohistochemistry and differences between groups were analysed using the Mann-Whitney U test. Immunostaining for IDO was located primarily in endothelial cell nuclei, with a reduced level of staining in the cytoplasm, in most capillaries from all placentae examined. A significantly higher level of IDO immunostaining was observed in normal placentae compared to pre-eclamptic placentae (P=0.008). 4-HNE was located mainly in the cytoplasm of syncytiotrophoblast cells of all placentae examined. There were no significant differences in the pattern or intensity of 4-HNE immunostaining levels between normal and pre-eclamptic pregnancies (P=0.684). Our IDO results support the hypothesis of decreased anti-oxidative capability in the placenta and the possibility of an ineffective compensatory mechanism against increased oxidative stress in the fetus.     

Evaluation of fetal serum thiol/disulphide homeostasis in deliveries complicated by nuchal cord

Aim: To investigate the serum thiol/disulphide homeostasis in deliveries complicated by nuchal cord (NC) and to compare the results with healthy deliveries (without NC).

Methods: This prospective controlled study included 48 pregnant women complicated by NC and 48 similar gestational aged healthy pregnant women during labor. Fetal umbilical cord serum samples were collected during labor and the thiol/disulphide homeostasis was measured by using an automated assay method. The patients were followed up until end of the delivery and perinatal outcomes were recorded.

Results: Fetal umbilical cord native thiol, total thiol, and disulphide levels as well as disulphide/native thiol and disulphide/total thiol ratios are impaired in labor with the presence of NC. There were no statistically significant differences in terms of maternal and gestational age at delivery and maternal number of gravida and parity, fetal gender, fifth Apgar scores <7, mode of delivery and fetal birth weight between groups. The group of patients with NC had higher emergency C/S numbers indicated for fetal distress and lower first Apgar scores <7. There were no neonatal intensive care unit admissions among these babies.

Conclusions: Maternal serum thiol/disulphide homeostasis reflect transient effects of NC during labor regardless of labor type. Vaginal delivery can be safely and successfully performed in pregnancies complicated with NC.     

Serial plasma fibronectin levels in pre-eclamptic and normotensive women.

OBJECTIVE: Endothelial cell damage has been put forward as an underlying factor for development of pre-eclampsia. This study was carried out to see if fibronectin, which is a marker of endothelial damage, could be used as a marker of pre-eclampsia. METHODS: A longitudinal study was conducted on 100 normotensive primigravidae registered before 20 weeks of gestation. These subjects were followed until delivery and three blood samples were collected, first at registration, i.e. before 20 weeks, second around 28 weeks and third at 36 weeks or later till delivery. Fibronectin levels were assayed by ELISA and women observed for any signs of pre-eclampsia. RESULTS: Fourteen subjects developed pre-eclampsia. Fibronectin levels were observed to rise as pregnancy advanced but the rise was significantly higher in subjects who developed pre-eclampsia. The fibronectin levels were also significantly higher in these 14 subjects even in the first sample, i.e. before 20 weeks of gestation when compared with normotensive subjects (P < 0.01). CONCLUSIONS: Fibronectin levels could be used as an early valuable biomarker for the development of pre-eclampsia.     

VEGF mRNA levels in placentae from pregnancies complicated by pre-eclampsia

Objective To measure the mRNA levels of vascular endothelial growth factor and its receptor in the placenta following delivery after uncomplicated pregnancy and after pregnancies complicated by pre-eclampsia.
Setting Rosie Maternity Hospital, Cambridge.
Material Placental biopsies were obtained following delivery by caesarean section in 23 cases of pregnancy presenting at a range of gestational ages with pre-eclampsia. These were compared with biopsies from 20 appropriately matched women with uncomplicated pregnancies.
Main outcome measure mRNA levels of vascular endothelial growth factor (VEGF) and its receptor the fins-like tyrosine kinase (flt), were quantified in total RNA isolated from placental biopsies using the RNAse protection assay. The amount of RNA was compared with that of the housekeeping gene glyceraldehyde 3–phosphate dehydrogenase (GAPDH), used as a standard. Results were expressed as arbitrary optical density units of VEGF/GAPDH and flt/GAPDH.
Results In both control and pre-eclamptic women regression analysis showed that the level of mRNA encoding VEGF declined significantly with gestational age (   P < 0.0001  ). However, levels of VEGF mRNA were significantly lower in the pre-eclamptic women compared with the control women (   P < 0.023  ).
Conclusions This study provides evidence of an abnormality of growth factor expression in the placenta during pregnancies complicated by pre-eclampsia. Such placentae exhibit deficient growth and differentiation of terminal villi and reduced fetal capillary branching and reduced levels of VEGF could well account for these morphometric changes. This finding provides a molecular explanation for this abnormal placental development and points to VEGF as a factor in the aetiology of pre-eclampsia and its complications.     

An alternative method for measuring oxidative stress in intrahepatic cholestasis of pregnancy: thiol/disulphide homeostasis

Purpose: The aim of our study was to evaluate the oxidative stress (OS) in pregnant women with intrahepatic cholestasis of pregnancy (ICP) by evaluating thiol/disulphide homeostasis using an alternative technique.

Methods: A total of 57 pregnant women with ICP were compared with 50 gestational age and body mass index matched controls. A recently defined method was used for the measurement of plasma native-total thiol and disulphide levels. The independent two-sample t test, Mann–Whitney-U test, Chi-square test, binary logistic regression with backward elimination and receiver operating characteristic (ROC) curve was performed for statistical analyses.

Results: Pregnant women with ICP (n?=?57) versus controls (n?=?50) had significantly lower serum levels of native thiol (233.8?±?47.4?μmol/L vs. 308.5?±?51.7?μmol/L, p?p?p?p?p?Conclusions: To our knowledge, this is the first study in the literature exploring thiol/disulphide balance in ICP. We found that thiol/disulphide balance indicate OS in pregnant woman with ICP.     

Evaluation of thiol/disulphide homeostasis as a novel predictor testing tool of early pregnancy viability

Objective

To evaluate serum dynamic thiol/disulphide concentrations in patients with suspected missed abortion (MA) and to determine whether this ratio has a predictive role in the viability in these pregnancies.