Activated protein C resistance in normal and pre-eclamptic pregnancies

OBJECTIVES: A lower ratio in the classic activated protein C resistance (APC-R) test has been reported during pregnancy, which has been called 'acquired' APC-R. However, little is known about the cause of the lowered ratio, and whether or not there is a correlation with blood coagulation activation. The primary objective of our study was to determine changes in APC-R levels in each of the trimesters of normal pregnancy. The secondary objective was to confirm whether APC-R levels were lower in pregnancies complicated by pre-eclampsia than in a control group. Finally, this prospective study was performed to investigate the prevalence of APC-R among pregnant women and to elucidate its obstetric consequences. METHODS: We enrolled 35 healthy pregnant women and 47 pregnant women affected by pre-eclampsia in our study. The following laboratory tests were performed: prothrombin time, partial thromboplastin time, fibrinogen levels, antithrombin III, plasmatic fibronectin (as a marker of endothelial damage), haptoglobin (as a marker of intravascular haemolysis), a functional test for APC-R and analysis of factor V Leiden mutation by polymerase chain reaction. RESULTS: The activated protein C sensitivity ratio was lower in the pathological group than in the control group (p = 0.008 and p = 0.02, respectively). Plasmatic fibronectin was found to be higher in the pathological group than in the control group (p = 0.05). Finally, the overall prevalence of factor V Leiden mutation was 5.4%, i.e. 2/35 women (5.7%) in the control group and 3/47 women in the pathological group (6.38%). CONCLUSIONS: The APC ratio decreased after 20 weeks of gestation until week 42. This decrease was most pronounced in the third trimester, in which resistance was demonstrated in 34.2% of control group patients. In pre-eclampsia, we found a greater reduction of the APC ratio than in controls. We hypothesise that this is due to a decrease in the plasmatic levels of coagulation inhibitors and an increase in coagulatory factors.     

N-terminal parathyroid hormone-related protein levels in human intrauterine growth restricted pregnancies

BACKGROUND: N-terminal parathyroid hormone-related protein has a vital role in regulating cell growth and differentiation, uteroplacental vasodilatation, uterine muscle relaxation, and placental transport. These functions are compromised in intrauterine growth restriction. We aimed to investigate N-terminal parathyroid hormone-related protein concentrations in maternal, fetal, and neonatal plasma of intrauterine-growth-restricted and appropriate for gestational age pregnancies. METHODS: Plasma N-terminal parathyroid hormone-related protein levels were determined in 40 mothers and their 20 intrauterine-growth-restricted and 20 appropriate for gestational age singleton full-term fetuses and neonates on postnatal days 1 and 4. RESULTS: Fetal N-terminal parathyroid hormone-related protein levels were significantly lower in the intrauterine growth restriction group (b=1.166, 95%CI: 0.430-1.902, p=0.003) and correlated with the customized centiles of the infants (r=0.407, p=0.009). In the appropriate for gestational age group, neonatal day 1 N-terminal parathyroid hormone-related protein levels were significantly lower compared to maternal (p<0.001) and fetal (p=0.022) ones. Fetal and neonatal day 1 levels were significantly lower in males (b=-1.303, 95%CI: -2.508 to -0.097, p=0.036, and b=-0.802, 95%CI: -1.5 to -0.105, p=0.027, respectively). In the intrauterine growth restriction group, maternal N-terminal parathyroid hormone-related protein levels were significantly increased compared to fetal (p<0.001) and neonatal day 1 (p=0.001) levels. CONCLUSIONS: The reduced fetal N-terminal parathyroid hormone-related protein concentrations in intrauterine growth restriction may reflect compromised placental function and impaired fetal growth, suggesting that N-terminal parathyroid hormone-related protein may be involved in the pathogenesis of intrauterine growth restriction.     

Lymphocyte subsets in normal and pre-eclamptic pregnancies

Summary. Peripheral blood lymphocyte subsets in normal and preeclamptic pregnancies have been studied by using the monoclonal antibodies OKT3 (T cells), OKT4 (helper/inducer T cells) and OKT8 (suppressor/cytotoxic T cells). In addition the numbers of mononuclear cells bearing la and monocyte antigens have been assessed by using the monoclonal antibodies OKIal and OKM1. No significant differences were found between 10 normal pregnant and 10 non-pregnant subjects. Ten preeclamptic patients were studied and showed an increase in OKT4-positive helper cells. This was significant in terms of percentage of mononuclear cells but not the absolute numbers or the helper/suppressor (OKT4/OKT8) ratio.     

Chitotriosidase and YKL-40 in normal and pre-eclamptic pregnancies

OBJECTIVE: To compare macrophage activation in normal and pre-eclamptic pregnancies by determining YKL-40 concentration and chitotriosidase activity in maternal and cord serum. METHODS: In this prospective case-control study samples of maternal peripheral blood and umbilical venous blood were collected from 28 pre-eclamptic and 24 normotensive pregnant women and their newborns. YKL-40 concentration and chitotriosidase activity were determined by enzyme-linked immunoassay and fluorometry, respectively. RESULTS: Chitotriosidase activity in maternal and cord serum and YKL-40 concentration in cord serum were significantly higher in pre-eclamptic pregnancies (P<0.001), but there was no significant difference in maternal serum levels of YKL-40 between the case and control groups (P>0.05). There was a significant positive correlation between diastolic blood pressure and (1) chitotriosidase activity in both maternal and cord serum and (2) cord serum concentration of YKL-40 (r=0.61, r=0.84, and r=0.58, respectively). CONCLUSION: This study may be the first to demonstrate maternal and fetal macrophage activation in pre-eclampsia.     

Maternal peripheral blood leukocytes in normal and pre-eclamptic pregnancies

Objective To analyse activation of maternal peripheral blood leukocytes by flow cytometric measurements of intracellular free-ionised calcium of lymphocytes, granulocytes, and monocytes, separately.
Design Case-control study.
Setting High risk pregnancy service in a regional centre.
Material Samples from 10 women with Pre-eclampsia, 10 appropriately-matched women with normal pregnancy, nine multigravid normal women at mid-gestation selected as being least likely to demonstrate any tendencies towards Pre-eclampsia, and 11 healthy nonpregnant women of reproductive age were studied.
Methods Using flow cytometry, intracellular free ionised calcium ([Ca²⁺]_i) was estimated by loading the cells with Fluo-3 and measuring the changes in fluorescence intensity induced by free ionised calcium. After the basal levels were measured, the response of phagocytes to stimulation with n-formylmethionyl-leucyl-phenylalanine (fMLP) was determined.
Main outcome measures Basal [Ca²⁺]_i of peripheral blood leukocytes.
Results Median basal [Ca²⁺]_i was significantly increased in all three subsets of leukocytes—lymphocytes, granulocytes and monocytes in Pre-eclampsia—compared with the three control groups. Samples from both groups of women with normal pregnancy did not differ from those from nonpregnant women. The peak responses of monocytes to stimulation with 10 nmol fMLP were greater in samples from pre-eclamptic women, giving evidence of priming.
Conclusions Peripheral blood leukocytes are activated in Pre-eclampsia in terms of basal changes in the intracellular second messenger—free ionised calcium. Peripheral blood monocytes are primed to give greater responses after stimulation with fMLP.     

Fetal magnetoencephalographic mapping in normal and pre-eclamptic pregnancies.

This study investigates the fetal brain activity in normal and pre-eclamptic pregnancies. Measurements were performed by means of a superconducting quantum interference device (SQUID) in an electrically shielded room of low magnetic noise. The study was prospective. Ten pregnant women with pre-eclampsia and 11 healthy gravidae were included. All were preterm at 28 to 37 weeks' gestation. Biomagnetic signals (waveforms), recorded from the fetal brains in the frequencies 2-7 Hz, were expressed in terms of magnetic power spectral amplitudes: these were low in almost all normal pregnancies, and high in most pregnancies complicated with pre-eclampsia. The pictorial representation of the results in the form of iso-spectral amplitude (ISO-SA) mapping showed two different patterns: (a) iso-contour lines 'organised' in dense concentration zones (pre-eclamptic pattern), (b) iso-contour lines at random distribution without dense concentration zones (normal pattern). Biomagnetic measurements of fetal brain activity is a promising screening procedure for assessing the cerebral function, especially at high risk pregnancies.     

Maternal peripheral blood leukocytes in normal and pre-eclamptic pregnancies.

OBJECTIVE: To analyse activation of maternal peripheral blood leukocytes by flow cytometric measurements of intracellular free-ionised calcium of lymphocytes, granulocytes and monocytes, separately. DESIGN: Case-control study. SETTING: High risk pregnancy service in a regional centre. MATERIAL: Samples from 10 women with pre-eclampsia, 10 appropriately-matched women with normal pregnancy, nine multigravid normal women at mid-gestation selected as being least likely to demonstrate any tendencies towards pre-eclampsia, and 11 healthy nonpregnant women of reproductive age were studied. METHODS: Using flow cytometry, intracellular free ionised calcium ([Ca2+]i) was estimated by loading the cells with Fluo-3 and measuring the changes in fluorescence intensity induced by free ionised calcium. After the basal levels were measured, the response of phagocytes to stimulation with n-formylmethionyl-leucyl-phenylalanine (fMLP) was determined. MAIN OUTCOME MEASURES: Basal [Ca2+]i of peripheral blood leukocytes. RESULTS: Median basal [Ca2+]i was significantly increased in all three subsets of leukocytes--lymphocytes, granulocytes and monocytes in pre-eclampsia--compared with the three control groups. Samples from both groups of women with normal pregnancy did not differ from those from nonpregnant women. The peak responses of monocytes to stimulation with 10 nmol fMLP were greater in samples from pre-eclamptic women, giving evidence of priming. CONCLUSIONS: Peripheral blood leukocytes are activated in pre-eclampsia in terms of basal changes in the intracellular second messenger--free ionised calcium. Peripheral blood monocytes are primed to give greater responses after stimulation with fMLP.     

Glutathione S-transferases and esterases in placenta after normal and pre-eclamptic pregnancies

Severe pre-eclampsia reduced significantly (P<0.05) by 68+/-6 per cent (mean+/-sem, n=10) the maximal velocity (V(max)) and, consequently, reduced significantly by 60+/-7 per cent the catalytic efficiency (C(E)) of placental glutathione transferase pi, assayed with ethacrynic acid. Mild and severe pre-eclampsia reduced significantly by 82+/-5 per cent (mean+/-sem, n=5) and by 41+/-5 per cent (mean+/-sem, n=10), respectively, the V(max)and, consequently, reduced significantly by 72+/-7 and by 33+/-13 per cent, respectively, the C(E)of esterase, assayed with p-nitrophenyl acetate. Furthermore, severe pre-eclampsia increased significantly by 296+/-78 per cent the Michaelis-Menten constant (K(m)) of total GST, assayed with chlorodinitrobenzene and, consequently, decreased significantly the C(E)by 83+/-3 per cent. On the other hand, the concentrations of total and non-protein thiols did not change significantly in placental homogenates from patients with mild or severe pre-eclampsia compared to normal pregnancies. These findings would indicate a decreased capacity of the glutathione transferases and esterase detoxification systems to protect the fetus from drugs prescribed to pregnant women suffering pre-eclampsia, mainly in the severe phase.     

Gestational effects on host inflammatory response in normal and pre-eclamptic pregnancies

Objective

Pre-eclampsia (PET) remains a leading cause of maternal and neonatal morbidity and mortality. Although its pathophysiology involves an underlying inflammatory dysfunction, it is unclear how this may be affected by increasing gestational age, particularly in relation to the time of onset of disease. Murine studies have indicated that a progressive increase in serum inflammatory profile is a physiological feature of normal gestation. The present study aimed to investigate this phenomenon in women in relation to normal and pre-eclamptic pregnancies.

Study design

Control and PET groups (each n = 20) were divided into early and late pregnancy (before and after 34 weeks gestation, respectively). Whole blood was diluted 1:1 with RPMI 1640 medium with/without 1 μg/ml lipopolysaccharide at 37 °C for 24 h under a humidified 5% CO₂ atmosphere. Samples were collected at 0, 2, 6 and 24 h and analysed for interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-γ, monocyte chemotactic protein (MCP-1), macrophage inflammatory protein (MIP)-1β and tumour necrosis factor (TNF)-α by fluid-phase multiplex immunoassay.

Results

This study confirms that pregnancy features an increasing inflammatory response with advancing gestational age, which was seen in both control and PET pregnancies (P < 0.01).

Conclusions

This increase in inflammatory responsiveness with advancing gestation may provide an explanation for the incidence of late onset PET in the absence of placental pathology, as well as serving as a potential physiological priming mechanism geared towards increasing maternal sensitivity to the fetal triggers of labour.     

Developmental aspects of parathyroid hormone-related protein biology

Parathyroid hormone-related protein (PTHrP) has been discovered as a parathyroid hormone (PTH)-like factor responsible for the humoral hypercalcaemia of malignancies. Further studies revealed that PTHrP is ubiquitously expressed, in mature as well as in developing normal tissues from various species. Although not completely understood, the biological roles of PTHrP concern a variety of domains, including calcium phosphorus metabolism and bone mineralization, smooth muscle relaxation, cell growth and differentiation, and embryonic development. As a poly-hormone, PTHrP is now acknowledged to act via the paracrine, autocrine, and even the intracrine pathways. This review focuses on the main developmental features of the biology of PTHrP. During embryonic development, PTHrP is considered to be involved as a growth factor that promotes cell proliferation and delays cell terminal maturation. PTHrP has been shown to intervene in the development of various tissues and organs such as the skeleton, skin, hair follicles, tooth, pancreas, and the kidney. In addition, through its midregion sequence, which is able to promote an active transplacental calcium transport, PTHrP may intervene indirectly in the mineralization of the foetal skeleton. PTHrP has also been shown to be necessary for the normal development of the mammary gland, while huge amounts of PTHrP are found in the human milk. Finally, observations of physiologic, vasodilating effects of PTHrP in the kidney suggest its involvment in the control of renal hemodynamics, especially in the perinatal period.     

SQUID biomagnetometry of the uterine arteries in normal and pre-eclamptic pregnancies.

AIM: This study was designed to investigate the hemodynamics of the uteroplacental circulation in normal and pre-eclamptic pregnancies using the biomagnetometer SQUID. METHOD: Twenty-two pregnancies complicated by pre-eclampsia and 49 normal pregnancies were included in this study. All were near term. Biomagnetic signals were recorded from the uterine arteries. After statistical Fourier analysis, the findings were designated in terms of spectral amplitudes as high (140-300 fT/square root of Hz), low (50-110 fT/square root of Hz) and borderline (111-139 fT/square root of Hz). RESULTS: The uterine artery waveforms and the corresponding spectral densities were of high amplitudes in most (89.7%) normal pregnancies and of low amplitudes in most (81.8%) pregnancies complicated by pre-eclampsia (p < 0.005). These findings were of statistical significance and were correlated with fetal heart rate (FHR) monitoring, pH, Apgar score at 1 and 5 minutes and birth weight percentiles: high amplitude cases were related with normal FHR patterns, pH > 7.25, Apgar score > 7 and birth weight > 75th percentile, while low amplitude recordings were connected with abnormal FHR patterns, pH < 7.25, Apgar score < 7, and birth weight < 10th percentile (8 cases) and < 50th percentile (10 cases). CONCLUSION: Biomagnetic measurement of the uterine artery flow, is a promising procedure in assessing fetal health, especially in high-risk pregnancies.     

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.     

Rescue of trophoblast apoptosis by parathyroid hormone-related protein

The effects of the 1–34 N-terminal parathyroid hormone-related protein fragment (0–10μM) and the 67–86 parathyroid hormone-related protein fragment (0–10μM) on trophoblast apoptosis, induced by TNFα and IFNγ or by the protein kinase inhibitor staurosporine, were investigated. TNFα/IFNγ and staurosporine significantly increased the rate of apoptosis by fivefold and by eightfold, respectively. Parathyroid hormone-related protein (1–34) evoked a dose-dependent rescue of both TNFα/IFNγ-induced and staurosporine-induced apoptosis, whereas parathyroid hormone-related protein (67–86) had no significant effect on staurosporine-induced apoptosis, and only significantly diminished TNFα/IFNγ-induced apoptosis at 10μM. Parathyroid hormone-related protein was thus found to be a cytotrophoblast apoptosis survival factor.     

Serum cystatin C reflects glomerular endotheliosis in normal, hypertensive and pre-eclamptic pregnancies

Objective To study the correlation between serum cystatin C levels and renal structural changes in normal, hypertensive and pre-eclamptic pregnancy to evaluate it as a marker of the degree of renal involvement in pre-eclampsia.
Design An observational prospective study.
Setting University Hospital of Lund, Sweden.
Sample Thirty-six women with hypertensive disease in pregnancy and 12 healthy pregnant women in the third trimester recruited from maternal health care centres in the catchment area of the hospital.
Methods Renal biopsy samples were obtained from all participants and the degree of endotheliosis as well as the mean glomerular volume was evaluated by light microscopy in silver methenamin-stained sections. Serum cystatin C levels were measured and correlated to the structural changes.
Main outcome measures Correlation among degree of glomerular endotheliosis, glomerular volume andserum cystatin C.
Results Serum cystatin C levels differed between the different degrees of endotheliosis, showing a highly significant increasing linear trend. They also correlated significantly with glomerular volume (   r = 0.60, P < 0.001  ). Mean serum urate and creatinine levels also increased with the degree of endotheliosis, but not above the reference interval for normal term pregnancy, even in pre-eclamptic women.
Conclusion Serum cystatin C may be used as a marker, not only for impaired renal function, but also for the degree of glomerular endotheliosis and increase in glomerular volume in pregnancy. It may be of value in the monitoring of pregnancies complicated by pre-eclampsia.