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Sildenafil citrate may offer a potential therapeutic strategy to improve uteroplacental blood flow in FGR pregnancies.

FETAL GROWTH RESTRICTION (FGR), when the fetus fails to attain its growth potential, affects approximately 8% of all pregnancies contributing to infant prematurity ( 1). FGR is responsible for considerable perinatal mortality and morbidity; infants are at increased risk of perinatal complications such as fetal distress, asphyxia, neonatal encephalopathy, hypothermia, hypoglycemia, and poor feeding as well as risks of long-term neurological and developmental disorders ( 2, 3).

These direct effects of FGR have massive implications for the delivery of health care ( 4). Infants of FGR pregnancies may show modification of metabolic/hematological pathways and altered immune function ( 5). In the long term, deficient growth in utero is related to an increased incidence of hypertension, diabetes mellitus, and coronary heart disease in adulthood ( 6– 8). The underlying pathology of FGR is multifactorial and poorly understood.

For example, growth restriction has been shown to have a genetic component such as trisomies and cases of placental mosaicism ( 9) or be a result of physiological factors such as maternal undernutrition ( 10, 11) or smoking during pregnancy ( 12). However, the reduced growth of the FGR fetus is typically associated with placental insufficiency, i.e .

abnormalities in placental growth, development, and function.

In normal pregnancy (NP), uterine blood flow increases dramatically from approximately 50 ml/min (8.3 ? 10 ?4 liter/sec) at 10 wk gestation to over 1200 ml/min (0.02l liter/sec) at term ( 13); this massive increase in flow is facilitated by a radical trophoblast-driven modification of the uterine spiral arteries ( 14). Using the noninvasive technique of Doppler

ultrasound

A poorly modified uteroplacental vasculature is also a hallmark of the current pathogenic model of preeclampsia ( 18, 19) where aberrant spiral artery modification leads to a hypoperfused uteroplacental unit later in pregnancy ( 20, 21). This results in the release of a factor(s) from the placenta that activates the maternal vascular endothelium.

These data suggest that the pathophysiology of FGR and preeclampsia may be related.

A recent report that markers of endothelial dysfunction are increased in FGR ( 22), a commonly reported phenomenon of preeclampsia, supports such a hypothesis ( 23, 24). Endothelium-dependent responses in a variety of blood vessels from women with preeclampsia are markedly different, compared with those isolated from NP women ( 25– 27).

We have demonstrated, using small-vessel myography, reduced endothelium-dependent responses to bradykinin in myometrial resistance arteries isolated from women with preeclampsia ( 26, 27) and have shown that the small residual response to bradykinin is entirely mediated via nitric oxide ( 28). The first aim of the current study was to investigate our hypothesis that uterine resistance artery function is aberrant in FGR, by performing the first comparison of myometrial small-artery vasoconstriction and endothelium-dependent relaxation in pregnancies complicated by FGR with that seen in NP.

Animal studies suggest that phosphodiesterase-5 (PDE5) inhibitors, such as sildenafil citrate, may improve uterine blood flow via cGMP-mediated endothelial relaxation of uterine vessels ( 29).

Our second aim was thus to assess the effects of PDE5 inhibition in small arteries from women whose pregnancies were complicated by FGR. Mary’s Hospital, Manchester, gave approval for this work, and consent was obtained for all tissue samples used in the study. The investigation conforms to the principles outlined in the Declaration of Helsinki ( 30). Myometrial biopsies were obtained at delivery by cesarean section from the upper lip of the lower segment incision from healthy women (NP; n = 27) with uncomplicated pregnancies.

Women with hypertension, diabetes, or other significant medical disorders were excluded. Biopsies were also obtained from women with intrauterine growth restriction (FGR; n = 12).

All women in this group were identified on the basis of serial antenatal ultrasound scans demonstrating deficient fetal growth and delivered by cesarean section for maternal or fetal reasons; the diagnosis of FGR was confirmed by an individualized birthweight ratio (IBR) found to be less than the fifth centile.

IBR is a correction of birthweight for known confounding variables and relates to a predicted birthweight calculated using independent coefficients for gestation at delivery, fetal sex, parity, ethnic origin, maternal height, and booking weight. The IBR enables a more accurate prediction of pregnancies with a poor outcome than birthweight for gestational age alone ( 31). General chemicals were obtained

from

The thromboxane mimetic U46619 was obtained from Calbiochem (Nottingham, UK). The PDE5 inhibitors sildenafil citrate (Viagra) and UK-343664 were obtained from Pfizer (Sandwich, UK). Biopsies were placed directly into ice-cold physiological salt solution, a modified Krebs solution (see below for chemical composition). Myometrial small arteries were identified and dissected under a stereomicroscope. Myometrial artery diameters were 379 ± 4 ?m for NP biopsies (n = 49 arteries from 27 patients) and 310 ± 2 ?m for FGR biopsies (n = 62 arteries from 12 patients).

Myometrial small arteries were carefully cut into small (approximately 2 mm) lengths and mounted onto 40-?m steel wire in a Danish Myo Technology (Aarhus, Denmark) M610 wire myograph as described elsewhere ( 32).

Myometrial arteries were normalized to 0.9 of L 13.3kPa using the classical normalization procedure ( 33) as we have described in detail elsewhere ( 32, 34). Initially, the bath contained 7 ml of physiological salt solution (in mmol/liter: 127.76 NaCl, 25 NaHCO 3 , 4.69 KCl, 2.4 MgSO 4 , 1.6 CaCl 2 , sildenafil tablet buy online 1.18 KH 2 PO 4 , 6.05 glucose, 0.034 EDTA; pH 7.4), warmed to 37 C and gassed with air/5%CO 2 .

Contraction/relaxation responses of myometrial small arteries.

Dose-response constriction curves to AVP (0.1–10 nmol/liter; 2-min intervals) or the thromboxane-mimetic U46619 (0.1–1000 nmol/liter; 2-min intervals) were constructed followed by relaxation to BK (0.1–1000 nmol/liter; 2-min intervals). Vessels were washed to baseline tension and then incubated for 1 h in the presence of 0, 10, or 100 nmol/liter of the PDE5 inhibitor sildenafil citrate or UK-343664.

The protocol of constriction to AVP or U46619/relaxation to BK was repeated as outlined above in the maintained presence of the PDE5 inhibitor.

Data were first tested whether they fitted to a Gaussian (normal) distribution using the Kolmogorov-Smirnov test. Data that passed the normality test were compared using parametric statistical tests (unpaired/paired t test; repeated-measures ANOVA) and are represented as

mean

The effect of PDE5 incubation on agonist-induced constriction of myometrial small arteries was compared by paired t test.

BK-induced relaxation of myometrial small arteries was compared using repeated-measures ANOVA. Data for the number of patients are indicated by (N).

Women whose pregnancies were complicated by FGR delivered significantly earlier in gestation than the NP group ( P TABLE 1. NP 27 30 (19–40) 2 (1–5) 1 (0–4) 123 (110–150) 75 (60–85) 38.9 (37.4–41.5)

3300

NP 27 30 (19–40) 2 (1–5) 1 (0–4) 123 (110–150) 75 (60–85) 38.9 (37.4–41.5) 3300 (2690–4110) 46 (7–88) FGR 12 28.5 (22–37) 3.5 (1–5) 1 (0–4) 120 (115–140) 81 (70–90) 37.9 (29.8–39.0) a 2460 (745–2860) b 3 (0–5) b.

Demographic and clinical details are given for NP women and women whose pregnancies were complicated by FGR.

All data are median with range in parentheses for maternal age at delivery, gravidity, parity, maximum attained blood pressure (BP), gestation at delivery, birthweight, and IBR. Groups were compared statistically using the Mann-Whitney U test. NP 27 30 (19–40) 2 (1–5) 1 (0–4) 123 (110–150) 75 (60–85) 38.9 (37.4–41.5) 3300 (2690–4110) 46 (7–88) FGR 12 28.5 (22–37) 3.sildenafil tablet buy online 5 (1–5) 1 (0–4) 120 (115–140) 81 (70–90) 37.9 (29.8–39.0) a 2460 (745–2860) b 3 (0–5) b. NP 27 30 (19–40) 2 (1–5) 1 (0–4) 123 (110–150) 75 (60–85) 38.9 (37.4–41.5) 3300 (2690–4110) 46 (7–88) FGR 12 28.5 (22–37) 3.5 (1–5) 1 (0–4) 120 (115–140) 81 (70–90) 37.9 (29.8–39.0) a 2460 (745–2860) b 3 (0–5) b.

Demographic

All data are median with range in parentheses for maternal age at delivery, gravidity, parity, maximum attained

blood

Groups were compared statistically using the Mann-Whitney U test. Small arteries from women whose pregnancies were complicated by FGR showed significantly increased vasoconstriction compared with that seen in small arteries taken from NP women ( P 0.05, t test).

Addition of AVP to myometrial small arteries from women with FGR evoked maximal constriction of 21.5 ± 0.4 kPa (n = 24 arteries from seven patients) compared with 16.7 ± 0.4 kPa (n = 22 arteries from seven patients) in NP women ( P = 0.06, t test).

However, the sensitivity of myometrial small arteries from women whose pregnancies were complicated by FGR to AVP-induced vasoconstriction was not significantly altered compared with vessels from NP women (EC 50 , 0.47 ± 0.15 nmol/liter in FGR vs .

These data suggest modified vasoconstriction of myometrial small arteries from women whose pregnancies are complicated by FGR.

Effect of U46619 and AVP on myometrial small arteries from NP women and women whose pregnancies are complicated by FGR. Data are expressed as active effective pressure production in kPa.

All data are mean ± sem ; the x axis shows concentration of agonist ? 10 ?x . ?, NP (n = 13, top ; n = 14, bottom ); ?, FGR pregnancy (n = 10, top ; n = 6, bottom ). Effect of U46619 and AVP on myometrial small arteries from NP women and women whose pregnancies are complicated by FGR.

Data are expressed as active effective pressure production in kPa.

All data are mean ± sem ; the x axis shows concentration of agonist ? 10 ?x .

?, NP (n = 13, top ; n = 14, bottom ); ?, FGR pregnancy (n = 10, top ; n = 6, bottom ). The effect of PDE5 inhibition on agonist-induced vasoconstriction is shown in Table 2.

In NP, myometrial small arteries incubated with 100 nmol/liter sildenafil showed decreased constriction to AVP ( P = 0.002, paired t test).

A similar trend was seen in vessels constricted with U46619, although this did not achieve statistical significance ( P = 0.14, paired t test). In women with pregnancies complicated by FGR, myometrial small arteries incubated with 100 nmol/liter sildenafil showed decreased constriction with either AVP or U46619 ( P = 0.002 and P = 0.04, respectively, paired t test).

Additionally, 10 nmol/liter sildenafil showed decreased constriction with AVP-constricted small arteries ( P TABLE 2. Effect of incubation with sildenafil citrate on agonist-induced constriction of myometrial small arteries.

U46619 0 10.6 ± 1.0 (7) 10.7 ± 0.9 0.97 23.6 ± 1.3 (10) 22.2 ± 1.2 0.43 10 14.9 ± 1.1 (7) 12.9 ± 1.3 0.06 17.3 ± 0.8 (7) 15.7 ± 0.8 0.16 100 15.5 ± 0.6 (13) 14.5 ± 0.7 0.14 19.4 ± 0.7 (13) 17.2 ± 0.7 0.002 AVP 0 17.8 ± 1.0 (7) 16.0 ± 1.2 0.24 22.7 ± 1.0 (7) 23.2 ± 1.0 0.33 10 14.0 ± 0.9 (7) 12.3 ± 1.1 0.22 16.5 ± 0.9 (7) 14.9 ± 1.0 sildenafil tablet buy online 0.04 100 16.7 ± 1.2 (8) 14.0 ± 1.1 0.002 24.1 ± 1.0 (10) 20.8 ± 1.8 0.05.

U46619 0 10.6 ± 1.0 (7) 10.7 ± 0.9 0.97 23.6 ± 1.3 (10) 22.2 ± 1.2 0.43 10 14.9 ± 1.sildenafil tablet buy online 1 (7) 12.9 ± 1.3 0.06 17.3 ± 0.8 (7) 15.7 ± 0.8 0.16 100 15.5 ± 0.6 (13) 14.5 ± 0.7 0.14 19.4 ± 0.7 (13) 17.2 ± 0.7 0.002 AVP 0 17.8 ± 1.0 (7) 16.0 ± 1.2 0.24 22.7 ± 1.0 (7) 23.2 ± 1.0 0.33 10 14.0 ± 0.9 (7) 12.3 ± 1.1 0.22 16.5 ± 0.9 (7) 14.9 ± 1.0 0.04 100 16.7 ± 1.2 (8) 14.0 ± 1.1 0.002 24.1 ± 1.0 (10) sildenafil tablet buy online 20.8 ± 1.8 0.05.

Data are expressed as active effective pressure production (kPa). Data at each concentration of PDE5 inhibitor were compared before and after PDE5 inhibitor using the paired Student’s t test.

Effect of incubation with sildenafil citrate on agonist-induced constriction of myometrial small arteries. U46619 0 10.6 ± 1.0 (7) 10.7 ± 0.9 0.97 23.6 ± 1.3 (10) 22.2 ± 1.2 0.43 10 14.9 ± 1.1 (7) 12.9 ± 1.3 0.06 17.3 ± 0.8 (7) 15.7 ± 0.8 0.16 100 15.5 ± 0.6 (13) 14.5 ± 0.7 0.14 19.4 ± 0.7 (13) 17.2 ± 0.7 0.002 AVP 0 17.8 ± 1.0 (7) 16.0 ± 1.2 0.24 22.7 ± 1.0 (7) 23.2 ± 1.0 0.33 10 14.0 ± 0.9 (7) 12.3 ± 1.1 0.22 16.5 ± 0.9 (7) 14.9 ± 1.0 0.04 100 16.7 ± 1.2 (8) 14.0 ± 1.1 0.002 24.1 ± 1.0 (10) 20.8 ± 1.8 0.05. U46619 0 10.6 ± 1.0 (7) 10.7 ± 0.9 0.97 23.6 ± 1.3 (10) 22.2 ± 1.2 0.43 10 14.9 ± 1.1 (7) 12.9 ± 1.3 0.06 17.3 ± 0.8 (7) 15.7 ± 0.8 0.16 100 15.5 ± 0.6 (13) 14.5 ± 0.7 0.14 19.4 ± 0.7 (13) 17.2 ± 0.7 0.002 AVP 0 17.8 ± 1.0 (7) 16.0 ± 1.2 0.24 22.7 ± 1.0 (7) 23.2 ± 1.0 0.33 10 14.0 ± 0.9 (7) 12.3 ± 1.1 0.22 16.5 ± 0.9 (7) 14.9 ± 1.0 0.04 100 16.7 ± 1.2 (8) 14.0 ± 1.1 0.002 24.1 ± 1.0 (10) 20.8 ± 1.8 0.05.

Data are expressed as active effective pressure production (kPa). Data at each concentration of PDE5 inhibitor

were

small

This relaxation was significantly attenuated in arteries from women whose pregnancies were complicated by FGR (residual constriction, 43 ± 8% in FGR vs .

30 ± 5% in NP; IC 50 , 84 ± 15 nmol/liter in FGR vs .

14 ± 9 nmol/liter in NP; P = 0.001, repeated-measures ANOVA). A similar relationship was seen with arteries constricted with AVP (residual constriction, 45 ± 12% in FGR vs .

80 ± 3% in NP; IC 50 , 21 ± 12 nmol/liter in FGR vs . 15 ± 7 nmol/liter in NP; P = 0.0009, repeated-measures ANOVA; Fig.

Endothelium-dependent relaxation of U46619-constricted myometrial small arteries.

Data are expressed as

percent

Top , Arteries from NP (?; n = 14) and arteries from FGR pregnancy (?; n = 16 vessels from 12 patients); bottom , arteries from FGR pregnancy, BK-only control (•; n = 10), and arteries from FGR pregnancy, BK with 100 nmol/liter sildenafil citrate (0; n = 10).

Data were compared before and after sildenafil by repeated-measures ANOVA. Endothelium-dependent relaxation of U46619-constricted myometrial small arteries. Data are expressed as percent maximal response to U46619. All data are mean ± sem ; x axis shows concentration of BK ? 10 ?x . Top , Arteries from NP (?; n = 14) and arteries from FGR pregnancy (?; n = 16 vessels from 12 patients); bottom , arteries from FGR pregnancy, BK-only control (•; n = 10), and arteries from FGR pregnancy, BK with 100 nmol/liter sildenafil citrate (0; n = 10).

Data were compared before and after sildenafil by repeated-measures ANOVA.

Endothelium-dependent relaxation of AVP-constricted myometrial small arteries.

Data are expressed as percent maximal response to AVP. All data are mean ± sem ; x axis shows concentration of BK ? 10 ?x .

Top , Arteries from NP (?; n = 14) and arteries from FGR pregnancy (?; n = 8); bottom , arteries from FGR pregnancy, BK-only control (•; n = 6), and arteries from FGR pregnancy, BK with 100 nmol/liter sildenafil citrate (0; n = 6). Data were compared before and after sildenafil by repeated-measures ANOVA. Endothelium-dependent relaxation of AVP-constricted myometrial small arteries.

Data are expressed as percent maximal response to AVP. All data are mean ± sem ; x axis shows concentration of BK ? 10 ?x . Top , Arteries from NP (?; n = 14) and arteries from FGR pregnancy (?; n = 8); bottom , arteries from FGR pregnancy, BK-only control (•; n = 6), and arteries from FGR pregnancy, BK with 100 nmol/liter sildenafil citrate (0; n = 6).

Data were compared before and after sildenafil by repeated-measures ANOVA. The effect of PDE5 inhibition on human myometrial small arteries was studied using U46619- and AVP-induced constriction.