Institute where work was conducted: Department of Obstetrics, Medicine and Renal Medicine, St George Hospital, and University of NSW, Kogarah, Sydney, NSW, Australia.
*Correspondence: Professor M. A. Brown, Department of Renal Medicine, St George Hospital, Kogarah, New South Wales 2217, Australia.
Objective 1. To characterise the forearm vascular reactivity of women with pre-eclampsia in the third trimester of pregnancy and compare it with that in normal or gestational hypertensive pregnancies. 2. To document female sex steroid (oestradiol, progesterone, oestriol and βhCG) levels in the three groups of women.
Design Forearm blood flow was measured by venous occlusion plethysmography during intra-arterial infusion of saline and vasoactive substances: angiotensin II, sodium nitroprusside, acetylcholine and NG-monomethyl-l-arginine (l-NMMA).
Setting Research laboratory at St George Hospital, Kogarah, Sydney, Australia.
Sample Fifteen non-pregnant women in the follicular phase of the menstrual cycle, 15 third trimester normal pregnant women, 13 women in the third trimester with gestational hypertension and 15 women with pre-eclampsia.
Main outcome measures Changes in forearm blood flow in response to vasoactive substances.
Results Normal pregnant women had higher baseline forearm blood flow than non-pregnant women, decreased vasodilator responses to sodium nitroprusside and reduced vasoconstrictor responses to angiotensin II. No difference in response to angiotensin II, sodium nitroprusside or l-NMMA was found among normal pregnant, pre-eclampsia or gestational hypertension women, but vasodilatory responses of pre-eclamptic women to acetylcholine were reduced compared with normal pregnant women. Higher serum progesterone levels were found in women with pre-eclampsia and gestational hypertension than in normal pregnancy.
Conclusion The hyperdynamic circulation of normal pregnancy is characterised by refractoriness to angiotensin II but this is not altered in pre-eclampsia. Pre-eclamptic women demonstrate a reduced vasodilator response to acetylcholine which, in the absence of any alteration in response to l-NMMA, implies that factors other than nitric oxide deficiency mediate the vasoconstriction of pre-eclampsia.
Altered vascular reactivity is an accepted feature of pre-eclampsia1 and it is assumed that peripheral vasoconstriction makes a substantial contribution towards the hypertension which accompanies this disorder. Whereas normal pregnancy is accompanied by an increase in cardiac output2, plasma volume3 and peripheral blood flow4 and a reduction in peripheral resistance and vascular tone, pre-eclamptic pregnancy features a reduced plasma volume compared with normal pregnancy5 and an increase in pressor response6.
Studies in animals7 and the peripheral vascular bed (human)8–10 support the hypothesis that nitric oxide is responsible, at least in part, for the hyperdynamic circulation of normal pregnancy. In rats, a pre-eclampsia-like syndrome can be induced by the administration of nitric oxide antagonists11, and can be reversed by the administration of nitric oxide substrate, l-arginine. However, a deficiency of nitric oxide has not been shown to contribute definitively to the pathogenesis of human pre-eclampsia12.
There are few in vivo studies of pregnant women examining response to specific vasoactive factors in resistance vessels. It is necessary to examine the response of resistance vessels (i.e. arterioles and small arteries) rather than the venous circulation as these vessels are mainly responsible for systemic vascular resistance. In a study of the forearm vascular bed in normal pregnancy using venous plethysmography, Anumba et al.8 demonstrated an increased vasoconstrictor response to nitric oxide blockade (NG-monomethyl-l-arginine [l-NMMA]) in normal pregnant women compared with non-pregnant women, but no difference between normal pregnant women and pre-eclamptic women. Williams et al.10, using the technique of venous occlusion plethysmography, measured greater vasoconstriction in the hand blood flow of first and third trimester normal pregnant women compared with non-pregnant women.
These studies argue for nitric oxide having a significant contribution to the peripheral vasodilatation in the forearm and hand of normal pregnant women but, unlike studies in animals, not necessarily in pre-eclampsia. We sought to extend this limited knowledge on vascular reactivity in human pregnancy by studying the forearm blood flow of normal pregnant women, those with gestational hypertension and those with pre-eclampsia. Our hypothesis was that forearm vascular reactivity would be increased in pre-eclampsia (as endothelial function is affected) but not in gestational hypertension. As factors other than nitric oxide are involved in vascular reactivity, we tested the vasoactive response to endothelium-dependent factors (l-NMMA and acetylcholine) and endothelium-independent factors (sodium nitroprusside and angiotensin II).
Fifteen normal non-pregnant women in the follicular phase of the menstrual cycle and 15 normal pregnant women in the third trimester were recruited within St George Hospital. All women (including the pregnant groups) were aged between 18 and 40 years. The study was discussed individually with the principal investigator and an information sheet was left with the woman who was telephoned or reviewed the following day to ascertain participation. Thirteen gestational hypertensive women and 15 pre-eclamptic patients were recruited either from the (outpatient) day stay unit or from the antenatal ward. The same consent procedure was undertaken as for the normal pregnant women.
Gestational hypertension was defined as sustained hypertension (an absolute value of greater than 140/90 mmHg) developing de novo in the second half of pregnancy. The diagnosis was confirmed by resolution of hypertension within 12 weeks postpartum and the absence of any other systemic complications, as described below.
Pre-eclampsia was defined as hypertension de novo as for gestational hypertension, but in addition, there had to be a systemic complication such as proteinuria (with urinary tract infection excluded) of at least 0.3 g/24 hours or by spot testing of the urine protein/creatinine ratio ≥30 mg/mmol13, renal insufficiency, liver disease, neurological disturbance or haematological disturbance (haemolysis or thrombocytopenia)14. In the pre-eclamptic group, all had significant proteinuria except one patient who had hypertension and new onset thrombocytopenia but who subsequently developed proteinuria after the study was conducted and before delivery.
Women attended the laboratory in a quiet, temperature-controlled (24–26°C) room. A light breakfast was allowed, but smoking and drinking alcohol or caffeine were prohibited for the 24 hours preceding the study. Women already receiving anti-hypertensives (oxprenolol or methyldopa) had this medication withheld for at least 8 hours prior to the study. A medical history and examination was conducted.
The women were weighed, height and forearm circumference (7 cm from the olecranon process) were measured. Baseline blood pressure and pulse were measured in a seated position after 10 minutes rest, three recordings were taken with a conventional mercury sphygmomanometer recording Korotkoff sound V for diastolic blood pressure. An average of these readings was recorded.
Blood was collected after a further 5 minutes rest for plasma concentrations of: oestradiol, progesterone, haemoglobin, renin, aldosterone, calcium, magnesium, creatinine, sodium, potassium and glucose (i.e. factors which may directly or indirectly be involved in vascular resistance).
Forearm blood flow was measured by strain gauge plethysmography in response to vasoactive factors. The method of plethysmography has been described by us15 and Webb16 previously. In brief, women lay with a left lateral tilt on a bed with both arms supported at 45° on pillows. The arms were at such a level as to be slightly above the right atrium. Inflatable cuffs were placed around both wrists and upper arms and the pressure of the wrist cuffs inflated above systolic pressure for a period of 3 minutes during each reading. The upper arm cuff was intermittently inflated to 40 mmHg to occlude venous outflow from the arm for 15 seconds at a time with 5 seconds rest.
A mercury in silastic strain gauge (Hokanson; Washington, USA) was placed at the widest part of both forearms (the unstretched circumference of the strain gauge being approximately 2 cm smaller than the circumference of the limb) and was connected to a plethysmograph (Vasculab SPG16; California, USA). Forearm arterial blood flow was derived from the change in forearm circumference measured by the strain gauge during inflation of upper arm cuffs16. Flow measurements were recorded approximately three times per minute using a data acquisition programme (BioPac AcqKnowledge 3.2).
A 27-gauge steel cannula was inserted into the left brachial artery under local anaesthesia to infuse substances into the isolated forearm, with venous return obstructed. The dose required for a local forearm effect is 100–1000 times smaller than that required for a systemic effect17. The dimensions of the needle were as small as possible to provide minimal interference with brachial flow. Normal saline was infused via the intrabrachial cannula as the control infusate at a constant rate of 1 mL per minute.
A randomised schedule of four vasoactive agents was then infused (apart from in the normal non-pregnant women where acetylcholine was not used). Each drug was infused at three incremental doses, non-pregnant women receiving an incrementally lower dose in view of their lower plasma volume: angiotensin II (Stanford Consulting Laboratory, Rydalmere, Australia; angiotensin II 2, 4, 8 ng/minute non-pregnant, 4, 8, 16 ng/minute pregnant), sodium nitroprusside (David Bull Laboratories, Victoria, Australia; sodium nitroprusside 0.25, 0.5, 1 μg/minute non-pregnant, 0.5, 1, 2 μg/minute pregnant), acetylcholine (‘Miochol’, CIBA Vision, Castle Hill, Australia; acetylcholine 3.25, 7.5, 15 μg/minute) and l-NMMA (Clinalfa, Switzerland 100, 200, 400 μg/minute non-pregnant, 200, 400, 800 μg/minute pregnant). l-NMMA was always the last substance infused because its effects upon the endothelium are known to last over 20 minutes18. Each dose of each drug was infused for 6 minutes: 3 minutes priming followed by 3 minutes while readings were taken. Saline was infused as a washout for a period of 21 minutes in between each drug. Simultaneous measurements of blood flow were made in the right (control) arm throughout the study and systemic blood pressure was monitored at the calf using a Critikon Dinamap (Johnson & Johnson, Sydney) automated machine.
The study was approved by the South Eastern Sydney Area Health Service Ethics Committee, and each woman participated voluntarily and gave written informed consent.
There is no consensus within the existing literature as to the best method of analysis of data from forearm plethysmography. Four main methods of analysis have been published: (1) a ratio method of left to right forearm blood flow which uses the percentage change in forearm blood flow between infused and control arms to express data graphically17; (2) a subtraction method of left to right forearm blood flow, using the difference in absolute values of forearm blood flow between arms to express data graphically19; both of these methods then compare data points with Repeated measures analysis of variance (RMANOVA) to account for the fact that observations over time in an individual may be correlated; (3) summary measures (in arbitrary units) of the total forearm blood flow response to different doses of drug can be compared8 (analogous to ‘area under the curve’ analysis in some literature); (4) the maximal response (i.e. the comparison of the maximal percentage change in forearm blood flow to infused drug in one group compared with the maximal response in another)8. For simplicity, we have chosen the methods that have been most published, viz. using a ratio of left to right arm forearm blood flow and summary measures of total forearm blood flow. RMANOVA was chosen as the most powerful method of analysis with a Greenhouse–Geisser correction to assess significance20. An unpaired Student's t test was used to compare normally distributed summary measures. Statistical significance was taken as P < 0.05.
Baseline forearm blood flows during normal saline infusion were normally distributed and were compared using ANOVA to ensure that baseline flow between drug infusions did not alter through the study. Comparisons were made between:
1Change in forearm blood flow during incremental dosages of infused drug (within-group RMANOVA);
2Forearm blood flow responses between the non-pregnant and normal pregnant women (between-groups RMANOVA);
3Forearm blood flow responses among the three groups of pregnant women (between-groups RMANOVA).
Baseline characteristics of the women are detailed in Table 1. There were no significant differences among the pregnant groups in age, weight or height. Blood pressure was, by definition, greater in the gestational hypertensive and pre-eclamptic groups compared with controls. The gestation the study was undertaken was slightly more advanced in the gestational hypertensive group (35 weeks) compared with controls (33 weeks), P= 0.031. There were two multigravid women in the pre-eclampsia group whose exclusion or inclusion made no difference to the statistical significance of the results.
Table 1. Demographic data for non-pregnant, normal pregnant, gestational hypertensive and pre-eclamptic women. Data are mean (SD).
Normal non-pregnant (n= 15)
Normal pregnant (n= 15)
Gestational hypertensives (n= 13)
Pre-eclamptics (n= 15)
*P < 0.05 vs normal pregnant women.
**P < 0.01 vs normal pregnant women.
***P < 0.001 vs normal pregnant women.
Gestation at study (weeks)
Gestation at delivery (weeks)
Systolic blood pressure at study (mmHg)
Diastolic blood pressure at study (mmHg)
Baseline forearm flow was greater in normal pregnant women, 10 (6) mL/100 mL/min, than in non-pregnant, 5 (1) mL/100 mL/min (P < 0.01). Although there was a trend for baseline flow to be lower in the pre-eclampsia and gestational hypertension groups, this was not significant; normal pregnant group average baseline flow 10 (6) mL/100 mL/min; gestational hypertension 8 (4) mL/100 mL/min; pre-eclampsia group 7 (3) mL/100 mL/min. This measurement was taken as an average of forearm blood flow during saline infusion at the commencement of the study in the infused (left) and control (right) arms.
Flow was measured in both arms at the commencement of the study and between each drug infused. Baseline flow was found to be consistent throughout the study for all groups and in both arms. Blood pressure, as measured in the calf, was also found to be consistent throughout the study for all groups.
Normal pregnant and non-pregnant women both vasoconstricted significantly during infusion of angiotensin II (P < 0.01 pregnant, P < 0.001 non-pregnant). There was significantly less vasoconstriction in the pregnant than in the non-pregnant group (P < 0.05). Additionally, the mean summated response (arbitrary units) to identical doses of angiotensin II was greater in non-pregnant women [−46 (26)] (unpaired t test P < 0.01) compared with pregnant women [−16 (33)] (Fig. 1).
In comparison of the normal pregnant, pre-eclampsia and gestational hypertension women, all groups exhibited a significant reduction in forearm flow with angiotensin II infusion (P < 0.01 normal pregnant, P < 0.001 gestational hypertension and P < 0.05 pre-eclampsia). There was no significant difference in response among the three groups using either method of analysis.
In all groups, there was a significant reduction in forearm flow with l-NMMA infusion (P < 0.01 pregnant and gestational hypertension, P < 0.05 non-pregnant and pre-eclampsia). There was no significant difference in response to l-NMMA between pregnant and non-pregnant women, or between the normal and hypertensive pregnant women using either method of analysis (Fig. 2).
In all pregnant groups (acetylcholine was not used in the non-pregnant women), there was a significant increase in forearm flow during infusion of acetylcholine (P < 0.001 normal pregnant, P < 0.01 gestational hypertension and pre-eclampsia) and there was a significantly greater response to acetylcholine in pregnant women than in gestational hypertensive or pre-eclamptic women (P < 0.05). Using summary measures to compare identical doses, the greater vasodilation of normal pregnant [472 (564)] vs pre-eclamptics [159 (165)] remained significant: P= 0.05, but the comparison of normal pregnant vs gestational hypertensives [264 (243)] was not: P= 0.23 (Fig. 3).
In all groups, there was a significant increase in forearm flow with sodium nitroprusside infusion (P < 0.001 for all). There was a significant difference in response to sodium nitroprusside between pregnant and non-pregnant women (RMANOVA P < 0.01). This was also apparent using a summary measures response, comparing identical doses, non-pregnant women (unpaired t test P < 0.01) had a significantly greater increase in forearm blood flow with sodium nitroprusside [238 (179)] than normal pregnant women [93 (80)] (Fig. 4).
There was no significant difference in response to sodium nitroprusside among the three pregnant groups using either method of analysis.
Serum creatinine levels were significantly higher in pre-eclamptic women than in normal pregnant women (Table 2). Serum albumin concentrations were significantly lower in both the gestational hypertension and pre-eclampsia groups compared with normal pregnant women, as were plasma renin concentrations. The platelet count of pre-eclamptic women was significantly lower than that of both the gestational hypertension and the normal pregnant groups (P < 0.05 both groups). Although the mean aldosterone/renin ratio was higher in the pre-eclamptic group than in the normal pregnant group, there was one outlier causing this difference. The mean ratio with her result removed was 551 (555), which did not differ significantly from the normal pregnant group. The aldosterone/renin ratio was significantly greater in the gestational hypertension group (Tables 2 and 3).
Table 2. Biochemical and haematological parameters. Data are mean (SD).
Normal non-pregnant (n= 15)
Normal pregnant (n= 15)
Gestational hypertensives (n= 13)
Pre-eclamptics (n= 15)
*P < 0.05 vs normal pregnant group.
**P < 0.01 vs normal pregnant group.
***P < 0.001 vs normal pregnant group.
†P < 0.05, pre-eclamptic group significantly different from gestational hypertensive group.
Platelet count (109/L)
Table 3. Endocrinological parameters.
*P < 0.05, significantly different from normal pregnant group.
**P < 0.01, significantly different from normal pregnant group.
***P < 0.001, significantly different from normal pregnant group.
Aldosterone/ renin ratio
1335 (3079), see text
We have examined forearm blood flow in normal, gestational hypertensive and pre-eclamptic third trimester women. Baseline flow was higher in normal pregnant than in non-pregnant women, confirming the peripheral hyperdynamic circulation of pregnancy. While this may be a potential confounding factor, we attempted to address the issue by administering an incrementally higher dose of drug to pregnant than to non-pregnant women, we acknowledge that the effects of shear stress have not been controlled for, but did not wish to add confounding factors of pre-constriction to the physiological environment of the forearm. Our study does not confirm work from previous studies showing that forearm vascular responses to nitric oxide synthase inhibition are enhanced in pregnancy, nor did we demonstrate any difference in response to nitric oxide inhibition between hypertensive and normal pregnancy. By contrast, this study does substantiate work from human isolated vessels21,22 that vascular reactivity to endothelium-dependent vasodilators (in particular acetylcholine) is impaired in pre-eclampsia. Additionally, a far greater percentage vasodilator response to acetylcholine than to sodium nitroprusside was seen in normal pregnant women (Figs 3 and 4), whereas the percentage responses to sodium nitroprusside and acetylcholine between gestational hypertensive and pre-eclamptic groups were similar. This implies that normal pregnant women have the capacity to further vasodilate an already vasodilated peripheral vascular bed through a mechanism specific to acetylcholine and not to sodium nitroprusside. Sodium nitroprusside acts as a nitric oxide donor producing vasodilation through its action on smooth muscle, so it is possible that in pregnancy the nitric oxide receptors of smooth muscle are ready saturated.
Although the action of acetylcholine on the endothelium in the past has been taken to indicate endothelium-dependent vasorelaxation via the nitric oxide pathway, acetylcholine effects are not specific to nitric oxide-dependent vasorelaxation. If further studies were entertained, more specific nitric oxide-dependent vasorelaxation might be produced via the use of bradykinin23 (which acts via the Gq protein24) histamine or the calcium ionophore A-23187. It is possible that the mechanisms of vasodilation of acetylcholine, which appear deficient in pre-eclampsia, are non-nitric oxide mediated (e.g. via PG12 or endothelium-derived hyperpolarizing factor). In contrast to this work, a study using a serotonin as a more specific endothelium-dependent stimulator of nitric oxide8, found a blunted response in both normal pregnant and pre-eclamptic women. Using the technique of forearm iontophoresis, no consensus of small vessel response to acetylcholine has been found. Eneroth-Grimfors et al.25 found no significant difference in laser Doppler measurment of blood flow responses to acetylcholine or sodium nitroprusside among non-pregnant, normal pregnant and pre-eclamptic women. In a recent study, Davis et al.26 found an increased response to acetylcholine in pre-eclamptic women, although the technique of iontophoresis is not so specific in targeting resistance vessels, as it measures those of the skin in addition to those of the muscle of the forearm.
A confounding factor to these studies that must be considered is the use of anti-hypertensive medication prior to forearm plethysmography. Drugs such as β-blockers can theoretically affect plasma renin activity, and therefore potentially vascular reactivity. However, in a longitudinal study of women with chronic hypertension in pregnancy, August et al.27 did not find that plasma renin activity was depressed from second to third trimester despite chronic treatment with β-blockers. In a study28 comparing non-pregnant essential hypertensive patients who were treated with those who were untreated, withholding medication prior to forearm plethysmography made no differences in forearm responses to acetylcholine and carbachol. Most of the gestational hypertensive group were taking medication prior to the study, most of the pre-eclamptic group were not, a maximum of 7 days treatment (and a minimum of one day) was received prior to the study. Because responses did not differ to infused agents according to whether the subject was taking medication or not, all results were included in the final analysis'.
This study was unable to confirm enhanced vascular responsiveness to angiotensin II by women with pre-eclampsia, which Gant et al.6 had shown by systemic infusion of angiotensin II. Although previous studies infusing angiotensin II intravenously at a dose of 8 ng/kg/min have produced a systemic increase in blood pressure, we found no difference in forearm vasoconstriction over a dose range of 2–16 ng/minute into the brachial artery. Although Anumba et al.29 reported a significant difference in response to angiotensin II between pre-eclamptic and normal pregnant women, there was considerable overlap of their data between normal and pre-eclamptic pregnancy, with their results achieving significance only on the summary measures response and not on maximal response. This may signify that if refractoriness to angiotensin II does exist in normal pregnancy, this is a result of decreased sensitivity within other vascular beds (e.g. the renal or uteroplacental circulations only). Alternatively, enhanced angiotensin II sensitivity may not be as universal a feature of pre-eclampsia as previously suspected. Kyle et al.30 studied 495 primigravid healthy pregnant women and found that the angiotensin II sensitivity test had a positive predictive value of only 19% at 28 weeks of gestation (although the negative predictive value was 87%), and similarly, other smaller studies31–35 found a lower positive predictive value than that demonstrated by Gant et al.6. It is also worth noting that the original observations of Gant et al.6 were made largely within teenage African-American pregnant women, who may have a different genetically determined response to vasoreactive substances than a European or Caucasian population.
Consideration should be given to different responses to angiotensin II between vascular beds, angiotensin II receptors may be affected by the pre-eclamptic process within the uteroplacental and renal beds, but perhaps not within the forearm. Two studies: one employing forearm venous plethysmography36 and one brachial artery Doppler flow37, showed that although non-pregnant essential hypertensive subjects had increased forearm resistance, the vascular reactivity of the brachial artery specifically towards angiotensin II (not to NA) was the same as in normal controls. Work in pregnant ewes38 suggests that the uterine but not systemic arteries are refractory to angiotensin II in pregnancy. Morgan et al.39 hypothesise that a local spiral artery renin–angiotensin system induces pregnancy remodelling, and that in pre-eclampsia, this mechanism is faulty (possibly by a genetic variation) within the uterine vascular bed.
Thus, it must be accepted that a limitation of this study is whether or not forearm vascular responses in pregnant women can be assumed to be predictive of systemic responses, and whether or not within the disease process of pre-eclampsia increased peripheral resistance is unique to some (e.g. the uteroplacental) or all vascular beds. The renal, splanchnic and uteroplacental vascular beds may be those that are most essential to determining peripheral resistance and those that are preferentially affected by the disease of pre-eclampsia. It is understandably difficult to study these beds in vivo. Alternatively, our findings would generally be in keeping with the concept of pre-eclampsia as a ‘crossover disease’ from high cardiac output–normal peripheral resistance disease in the preclinical phase to a low cardiac output–high peripheral resistance disease40 at a later stage.
We found no difference among the pregnant women in plasma levels of 17-β oestradiol, oestriol, human chorionic gonadotrophin or the oestradiol/progesterone ratio. We did document significantly higher levels of progesterone in pre-eclamptic and gestational hypertensive compared with normal pregnant women and it is unlikely that the two weeks increase in gestational age in the gestational hypertensive group would account for this. Previous studies comparing placentas from women with pre-eclampsia to those from normal pregnancy have shown that there was an increased production of progesterone in the pre-eclamptic group41,42; in his study, Walsh41 postulates that progesterone may be responsible for vasoconstriction by inhibiting placental prostacyclin production. On the other hand, early studies did not find an increase in serum progesterone in pre-eclamptics43 and more recent studies have failed to demonstrate any correlation with placental prostacyclin production44.
This study has confirmed a hyperdynamic circulation with a blunted vasoconstrictor response to angiotensin II in normal pregnancy. No difference was found in forearm blood flow among third trimester normal pregnant, gestational hypertensive and pre-eclamptic women in response to intrabrachial infusions of angiotensin II, sodium nitroprusside or l-NMMA. However, women with pre-eclampsia have a reduced vasodilator response to acetylcholine compared with normal pregnant women. This might imply a reduced nitric oxide response, but because the response to l-NMMA was not affected by pre-eclampsia, other mechanisms are likely to be responsible for this effect. Future studies should examine the role of other vasoactive factors and elucidate the number of pre-eclamptic women with high, normal or low cardiac output states.