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Keywords:

  • Pre-eclampsia;
  • pregnancy;
  • sildenafil

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

Please cite this paper as: Herraiz S, Pellicer B., Serra V, Cauli O, Cortijo J, Felipo V, Pellicer A. Sildenafil citrate improves perinatal outcome in fetuses from pre-eclamptic rats. BJOG 2012;119:1394–1402.

Objective  To evaluate perinatal outcome after sildenafil citrate (SC) administration at the onset of pregnancy in a rat pre-eclampsia model.

Design  In vivo animal experimental study.

Setting  Fundación IVI-Instituto Universitario IVI, Valencia, Spain.

Sample Control and pre-eclampsia-induced pregnant Wistar rats exposed to chronic SC administration.

Methods  We evaluated the use of SC, which was tested as a potential therapeutic tool to maintain vasodilatation in complicated pregnancies. We have demonstrated previously that SC shows a hypotensive selective effect in normal rat pregnancies when compared with nonpregnant animals.

Main outcome measures  Maternal blood pressure, weight and mortality during pre- and postnatal development, maternal blood cellularity and haemodynamic changes with maternal and fetal Doppler quantitative indices.

Results  SC restores normal values of blood pressure, cell count and proteinuria for maternal syndrome. In offspring, SC improves weight gain and increases survival rates without fetotoxic effects. According to the haemodynamic results, SC has a significant effect on the resistance index in the uterine artery in pre-eclamptic animals, as it restores normal values to correlate with an increase in fetal perfusion through the ductus venosus.

Conclusions  These results suggest that SC administration during pregnancy may have a potential benefit for the treatment of hypertension during pregnancy by reversing the maternal effects of pre-eclampsia and by improving uteroplacental and fetal perfusion.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

Pre-eclampsia (PE) is the most important cause of maternal mortality in developed countries, and of perinatal morbidity/mortality as a result of intrauterine growth restriction and prematurity. It is present in 2–8% of all pregnancies and causes almost 15% of maternal deaths in the USA. Moreover, women who have PE in their first pregnancy have a seven-fold risk of recurrence.1–3

There are numerous data to support the theory that PE is caused by failure of the early trophoblast invasion of the myometrial portion of the spiral arteries, which results in the persistence of their muscular wall, causing vasoconstriction and a restriction of the maternal blood flow to the placenta.4,5

Sildenafil citrate (SC) is a potent vasodilator that enhances and prolongs the action of cyclic guanosine monophosphate by selectively inhibiting phosphodiesterase-5 inhibitors. Phosphodiesterase-5 is present in the fetoplacental circulation in animals.6 SC produces vasodilatation in human chorionic plate arteries in vitro.7 Previous reports have demonstrated that SC may improve uterine blood flow via cyclic guanosine monophosphate-mediated endothelial relaxation of uterine vessels, and that it also causes relaxation of the human myometrium in a concentration-dependent manner.8–10 We have demonstrated the placental transfer of SC in a group of normotensive rats. We observed that SC appears to have a selective hypotensive effect at the onset of pregnancy, implying increased fetal blood supply, an effect that was not present in nonpregnant animal groups.11 Moreover, the cellular mechanisms by which the inhibitors of phosphodiesterase-5 improve endothelial function and cell survival in critical situations after ischaemia and reperfusion have been described.12 In in vitro terms, SC reduces vasoconstriction and significantly improves vasodilatation of small endometrial arteries in placental bed biopsies from pre-eclamptic women.13,14In vivo studies in pregnant women with severe early-onset fetal growth restriction have revealed that SC treatment is associated with increased fetal growth.15 Therefore, the SC molecule possesses certain features that make it appealing for the treatment of pregnant women with PE, and there is growing interest in the analysis of its effects on animals.

The aim of this study was to ascertain whether early SC administration during pregnancy is able to safely eliminate the effects of an animal PE model in pregnant rats and their offspring, and thus to improve outcome.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

Animals and study design

An animal PE model with hypertension, proteinuria and fetal growth restriction can be developed using a nonselective inhibitor of all isoforms of nitric oxide synthase (NOS) (N-nitro-l-arginine methyl ester, l-NAME).11,16–20

Female Wistar rats were maintained under similar conditions to those reported previously.11,16 Eighty-six pregnant rats were randomly allocated to four groups from gestational day E0. A control group of pregnant animals (n = 23) received food and mineral water ad libitum. A second group (n = 21), called the PE group, received 50 mg/kg/day of l-NAME dissolved in their drinking water. The third group of animals, the PE + SC group (n = 17), received 50 mg/kg/day of l-NAME supplemented with 4 mg/kg/day of SC dissolved in water. The last group of animals was the SC group (n = 25), and these animals received only 4 mg/kg/day of SC dissolved by sonication in their drinking water. Data from this SC group have been reported in part previously.11 This SC dose has been tested as being effective by our group and others.11,21–26 l-NAME and SC were prepared daily at 09.00 hour to ensure freshness, and to avoid evaporation and contamination. The amount of water (30 ± 5 ml) taken by each rat before the onset of treatment was determined by use of a metabolic cage, and a 24-hour urine sample collection allowed the detection of the presence of micro-albumin. Proteinuria, established by repeated dipstick testing (Combur-Test™, Boehringer Mannheim, Vienna, Austria), of 2+ or more was considered to correspond to proteinuria (>300 mg per 24 hours).27–29

Pregnancy in rats lasts between 22 and 23 days (E23), the second trimester begins on E9.5–10 and the third trimester on E16.30,31 Animals were sacrificed on gestational days E8 (n = 4), E11 (n = 4–5) and E18 (n = 4–9) to evaluate the effects on the fetuses on these gestational days, which correspond to early, mid and late pregnancy, respectively. The mothers remaining from each group (n = 5–6) were allowed to deliver. Treatment administration ended at the time of delivery. Pups were studied from postnatal day 1 (PND1) and were submitted to a 10-week post-delivery follow-up (PND70). Anaesthesia was used for ultrasound examinations, and was induced with 8% sevoflurane (Sevorane, Abbott Laboratories S.A., Madrid, Spain) and maintained with 1.5% sevoflurane applied by mask.

The study was performed in accordance with European Directive 86/609/EEC and the National Institutes of Health (NIH) Guidelines for the welfare and use of laboratory animals. The study protocol used was approved by the Ethics Committee of the Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain.

Systolic blood pressure (SBP)

We measured SBP values using a noninvasive automatic blood pressure analyser (NIPREM 564, Cibertec, Madrid, Spain) attached to the tail of rats after previously dilating vessels with a stove (36–37°C) for 5 minutes, as described elsewhere.11,32–34 The mean of five readings from each animal was considered to be the individual SBP value. Measurements were taken on gestational days E0, E7, E10 and E17 from 12 randomised rats per group.

Effect on fetal and placental tissues

Various approaches were followed to analyse the weight of the different structures. On gestational days E8 and E11, we recorded the weight of a section of the pregnant uterus between implantation sites, which included the fetus as well as the associated uterine walls.17 Each fetus and its placenta were weighed on day E18 and the crown–rump length was measured. The resulting weights were adjusted by the total number of embryos/fetuses and maternal weight. Fetal organs were macroscopically reviewed in five fetuses per group to rule out congenital anomalies. Offspring weight was measured on a weekly basis until PND70. SBP was measured in the offspring of all the groups when animals were adults (PND70), as described above for pregnant animals.

Intrauterine mortality was calculated on gestational days E8 and E11 by counting the number of resorptions, and on E18 by evaluating the number of atrophic fetuses in the uterine horns of each mother, as reported previously.11 The cumulative survival rate was evaluated until PND60.

Doppler ultrasound examinations

On gestational days E8, E11 and E18, the anterior abdominal surface of the anaesthetised pregnant Wistar rats was shaved and examined using a 13-MHz linear array transducer provided with a colour-coded Doppler system and a pulsed wave Doppler ultrasound (Vivid 7 PRO BT04, GE Medical Systems, Horten, Norway). Maternal uterine arteries (Figure S1) were insonated on gestational days E8, E11 and E18, as described previously.11,35 Three consecutive cardiac cycles with similar flow velocity waveforms were studied. The average pulsatility index (PI) of the right and left uterine arteries was used for further analyses. The ductus venosus (DV) was insonated in a subgroup of fetuses in the PE (n = 9), PE + SC (n = 6), SC (n = 8) and control (n = 13) groups on gestational day E18.11,35 The DV maximum flow velocities (Vmax) during the ventricular systole (S) and early ventricular diastole (D) and the end-diastolic velocity at atrial contraction (a) were calculated as the mean values of three consecutive cardiac cycles. The DV mean flow velocity (Vmean) was calculated as Vmean = 0.7Vmax.36 The DV venous pulsatility index (VPI = S – a/Vmean) and the S – a/S ratio were also studied.37 The angle between the Doppler ultrasound beam and the insonated vessel was always <30°.

Blood analysis

Maternal blood was obtained from tail veins on days E8, E11 and E18. Blood samples were mixed with 3.9% citrate buffer as an anticoagulant (one part buffer, nine parts blood) and immediately diluted (20×) with Tyrode buffer (pH 7.4), and supplemented with 0.55 m glucose and 3.5% bovine serum albumin (Sigma, St Louis, MO, USA). Afterwards, 50 μl of this dilution were added to a 12 × 75-mm2 polypropylene tube and mixed with 50 μl of fluorospheres (Flow-Count Fluorospheres, Beckman Coulter Inc., Brea, CA, USA) and 450 μl of Tyrode buffer. This mixture was tested in a Cytomics F500 MCL cytometer equipped with an ionic laser which emitted at 488 nm using CXP software (Beckman Coulter Inc.). Total blood cell and platelet counts were determined in the blood samples.

Statistical analysis

Numerical data were expressed as the mean ± standard error of the mean. Comparisons between multiple groups were performed using analysis of variance and a Newman–Keuls post hoc test. An unpaired t-test was performed when comparing two groups. Chi-squared or Fisher’s exact test was used to assess differences in proportions between study groups. Significant differences were considered when the probability value was <0.05. To estimate the surviving proportion, we followed the Kaplan–Meier method (for each time interval, we estimated the probability that rats that survived at the beginning would survive until the end). Two statistical software packages were used: GraphPad Prism 4.0. (GraphPad Software Inc., La Jolla, CA, USA) and SPSS 17.0 (SPSS Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

Maternal SBP and proteinuria

A significant increase in SBP was observed from the beginning of pregnancy in the PE group relative to the control group (E7, P = 0.033; E10, P < 0.0001; E17, P = 0.04). In the SC + PE group, SBP values remained similar to those of the control group throughout pregnancy (P = NS). Chronic SC administration produced a hypotensive situation during the first half of pregnancy, with significant decreases in SBP on E7 and E10 relative to the control group (P < 0.0001 and P < 0.0001, respectively) (Figure 1).

image

Figure 1.  Systolic blood pressure (mmHg) values throughout pregnancy in all groups. Values are shown as mean ± standard error of the mean, n. Significant differences from the control group are as follows: *P = 0.03, **P < 0.0001, ***P = 0.04, ****P < 0.0001 and *****P < 0.0001. PE, pre-eclampsia; SC, sildenafil citrate.

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A significant increase (P = 0.041) in the level of urine protein secretion was noted for the PE group from the beginning of pregnancy, with values higher than 300 mg in 24 hours. SC administration in the PE group reduced protein secretion to values below 300 mg, which also occurred in the control and SC groups, where animals always presented levels of urine protein secretion lower than 300 mg in 24 hours.

Perinatal outcome

Embryo/fetal weights were significantly lower in the PE and SC + PE groups relative to the control group on gestational days E8, E11 and E18. In contrast, fetal weights increased in the SC group relative to controls on days E11 and E18 (Table 1).

Table 1.   Embryo/fetal/litter and organ weights. The results are shown as mean ± standard error of the mean and sample size. Fetal and organ weights correlated with maternal and fetal weights, respectively
 ControlPEPE + SCSC
  1. CRL, crown–rump length; E, gestational day; PE, pre-eclampsia; PND, postnatal day; SC, sildenafil citrate.

  2. Statistically significant differences relative to the control group: *P < 0.01; **P < 0.0001; ***P < 0.05; ****P < 0.00001.

E8 fetal weight (g)0.084 ± 0.003, n = 340.066 ± 0.003, n = 27*0.069 ± 0.003, n = 45*0.089 ± 0.017, n = 43
E11 fetal weight (g)0.193 ± 0.007, n = 120.204 ± 0.008, n = 11*0.196 ± 0.018, n = 13*0.391 ± 0.044, n = 36***
E18 fetal weight (g)2.679 ± 0.113, n = 661.871 ± 0.040, n = 19**2.27 ± 0.170, n = 65*3.087 ± 0.112, n = 60***
PND1 weight (g)5.995 ± 0.093, n = 735.468 ± 0.070, n = 49**5.227 ± 0.049, n = 38*6.393 ± 0.187, n = 62
PND7 weight (g)11.85 ± 0.58, n = 209.92 ± 0.625, n = 12*11.10 ± 0.327, n = 2112.09 ± 0.30, n = 22
PND42 weight (g)132.0 ± 5.89, n = 30127.9 ± 3.089, n = 10128.16 ± 2.62, n = 21121.2 ± 6.01, n = 25
E18 CRL (cm)2.65 ± 0.08, n = 342.63 ± 0.22, n = 172.14 ± 0.05, n = 283.52 ± 0.03, n = 28
E18 placental weight (mg)649.4 ± 32.9, n = 12546.7 ± 22.7, n = 46***548.4 ± 36.6, n = 37***732.3 ± 28.4, n = 30****
E18 fetal liver/body weight0.030 ± 0.003, n = 250.023 ± 0.002, n = 140.020 ± 0.001, n = 240.067 ± 0.004, n = 35****

On PND1, the offspring weights from the PE and SC + PE groups were lower than those for the control animals. However, 1 week later (PND7), the offspring weight remained low only for the PE group. The weights of the SC group pups were similar to those of the control pups, except on PND1, when this weight increased. The placental weight on day E18 in the PE and SC + PE groups was lower than that for the control group. The placental weight was greater for the SC group (Table 1).

A macroscopic examination of the different organs and tissues revealed no anomalies, other than larger livers, in the SC group (P < 0.0001).

The number of live pups born per mother was slightly lower in the PE group than in the controls (PE, 8.1 ± 0.8 pups; controls, 12.1 ± 1.1 pups; P = 0.02). No significant differences were found in the other groups relative to the controls (SC + PE, 9.5 ± 2.1 pups; SC, 10.3 ± 1.4 pups).

A significant increase in intrauterine demise was noted on E18 (20% increase in fetal deaths per mother) in the PE group, and the number of fetuses was also lower (P = 0.03). In the PE + SC group, the number of resorptions on E18 was not significantly different relative to the control group (P = NS). The postnatal survival rate was lower for the PE (81.66%) and SC + PE (87.17%) groups relative to the control group (98.63%; P < 0.0001 and P < 0.05, respectively) (Figure 2).

image

Figure 2.  Survival rates until adulthood calculated by the Kaplan–Meier method. Descendents from the pre-eclampsia (PE) group have little probability of reaching maturity or adulthood. Sildenafil citrate (SC) administration increases survival in PE offspring, but does not control the offspring survival rate. Significant differences from the control group are as follows: *P = 0.01, **P = 0.0008.

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No differences were detected in SBP (control, 112.5 ± 2 mmHg; PE, 119.4 ± 3.8 mmHg; PE + SC, 115 ± 6 mmHg; SC, 118 ± 10 mmHg; P = NS) for offspring when taken in their adulthood (PND70) to elucidate whether maternal treatment had affected the blood pressure.

Fetal–maternal Doppler measurements

As shown in Table 2, the uterine artery PI was lower in the PE and SC groups on gestational day E8. On gestational day E11, the uterine artery PI was lower only in the PE group. Finally, the uterine artery PI was lower only in the SC group on gestational day E18.

Table 2.   Uterine artery pulsatility index (PI) and systolic peak velocity (SPV) determined by ultrasound scan in the uterine artery of rats throughout pregnancy. When compared with controls, PE animals displayed a decrease in uterine artery PI on gestational days E8 and E11.As reported previously, SC increased uterine artery PI on E8 and E18. SC administration in PE restores uterine artery PI and SPV to the values shown for the control group. No significant differences were found when SPV was compared among the groups
 Uterine artery PISPV (cm/seconds)
DayControlPEPE + SCSCControlPEPE + SCSC
  1. PE, pre-eclampsia; SC, sildenafil citrate.

  2. Statistically significant differences relative to the control group: *P < 0.0001; **P < 0.0001; ***P = 0.015; ****P = 0.014.

E81.43 ± 0.031.00 ± 0.09*1.46 ± 0.061.33 ± 0.02***11.1 ± 1.477.9 ± 1.67.15 ± 0.58.3 ± 1.63
E111.39 ± 0.030.93 ± 0.13**1.26 ± 0.071.37 ± 0.0815.0 ± 1.95.8 ± 1.28.4 ± 0.910.82 ± 2.49
E181.37 ± 0.031.27 ± 0.151.17 ± 0.151.37 ± 0.38****16.1 ± 1.89.7 ± 1.511.8 ± 3.416.8 ± 3.42

The individual analysis of uterine artery peak velocities showed a progressive increase in systolic (Table 2) and diastolic velocities during pregnancy for all groups, except the PE group (Figure 3).

image

Figure 3.  Uterine artery diastolic peak velocity of pregnant animals. The end-diastolic velocity (EDV) showed increased values throughout pregnancy in the control group. In the pre-eclampsia (PE) group, EDV decreased with pregnancy, showing the opposite pattern to the control group. This alteration was restored with sildenafil citrate (SC) administration in the PE + SC group, although EDV on E8 was lower than in the control group (*P < 0.0001). SC administration in normal pregnancy also produced lower EDV values on E8 (**P < 0.05), but increased values on E18 (***P = 0.016).

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The maternal heart rate in normal pregnancy increased significantly at the beginning of the second trimester (E11) and slowed down in the third trimester (E18) relative to previous values (E8, 384.5 ± 6.5 beats/minute; E11, 405.5 ± 6 beats/minute; E18, 203.5 ± 5 beats/minute; P = 0.004 and P < 0.0001, respectively). The PE group displayed a similar behaviour to the control group, but with an accentuated increase on E11 (E8, 168 ± 6 beats/minute; E11, 225 ± 9.5 beats/minute; P = 0.029) and a decrease on E18 (E18, 205 ± 8 beats/minute; P = 0.043). SC administration (PE + SC group) corrected these effects.

The DV VPI after chronic SC administration was lower in both the PE + SC and SC groups (control, 0.85 ± 0.02; PE + SC, 0.61 ± 0.08; P = 0.002; SC, 0.64 ± 0.02; P < 0.0001; PE, 0.89 ± 0.07; P = NS). The same differences were observed when the S – a/S ratio was analysed in both the PE + SC and SC groups, as shown in Figure 4A.

image

Figure 4.  (A) The S – a/S ratio of the fetal ductus venosus (DV). Sildenafil citrate (SC) administration brought about a significant decrease in the DV S – a/S ratio for the fetuses in the SC and pre-eclampsia (PE) + SC pregnant rats (control, 0.59 ± 0.01; SC, 0.48 ± 0.01; PE + SC, 0.46 ± 0.05; *P = 0.0002 and **P = 0.043, respectively). No differences were found in PE fetuses (PE, 0.58 ± 0.04, P = NS). (B) E18 DV atrial (a)-wave. Significant differences were found when the a-wave was individually analysed between the control and PE groups (control, 0.065 ± 0.003 cm/seconds; PE, 0.031 ± 0.005 cm/seconds; *P > 0.0001). SC administration recovered the a-wave to similar values to those recorded in the control group (PE + SC, 0.055 ± 0.005 cm/seconds; SC, 0.055 ± 0.001 cm/seconds; P = NS).

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The increased preload heart was evaluated by atrial (a)-wave measurement in the third trimester (Figure 4B). The velocity decreased significantly in the PE group (control, 0.065 ± 0.003 cm/seconds; PE, 0.032 ± 0.005 cm/seconds; P < 0.0001).

After SC treatment in the PE + SC group, this effect was corrected and no differences were found relative to the control group (PE + SC, 0.055 ± 0.005 cm/seconds).

Maternal peripheral blood cell count

A smaller total number of blood cells measured on E8 and a greater number on E11 were observed in the PE group relative to the control group, although the decrease was not significant. After chronic SC administration in the PE model, higher cellularity values relative to the control were obtained on E8, which became significant on E11 (P < 0.0001).

Platelet concentration increased progressively during pregnancy in the PE group, whereas the opposite effect was noted in the control group. In the PE + SC group, chronic SC administration was able to bring platelet levels back to normal values in the third trimester.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

Our study shows the effects of chronic SC administration from the beginning of pregnancy in an animal PE model. Our data suggest that SC is able to reverse the effects of an animal PE model in mothers and fetuses with similar perinatal outcomes to normotensive pregnant rats. SC is not associated with any increase in maternal or fetal mortality rates.

A previous study using the same PE model administered SC at a later stage in pregnancy and used higher SC doses.26 We believe that the administration of SC at an earlier stage in pregnancy is crucial to act on placentation development by completely reversing the PE-induced changes. Our results demonstrate that SC-treated pregnant rats maintain a similar blood pressure pattern to that of control rats throughout pregnancy. Another study that initiated SC therapy on day 7 showed that treatment was associated with lower maternal arterial pressure values, but normality was not achieved.19. The mechanism by which SC may cause this effect is unknown. It has been shown that the administration of SC to pre-eclamptic rats, induced with l-NAME, causes a decrease in the plasma levels of the anti-angiogenic factors endoglin (sEng) and fms-like tyrosine kinase1 (s-Flt-1).20 This finding suggests that the effect of SC may be related to vasodilatory substances that act locally in the placenta. In addition, it is known that SC has pro-angiogenic properties and is useful for the reperfusion of cerebral and heart tissues after the induction of hypoxia in animal models.38–40 Our group has shown previously that SC is able to promote the differentiation of neurons from stem cells in fetuses with induced hypoxia using the same animal PE model by administering the same SC dose, which proved to be a safe and effective measure.11,23,24 Therefore, SC seems to be an ideal candidate for acting on not only the initial placenta, which develops under hypoxic conditions at the beginning of pregnancy, but also on the cerebral development of compromised fetuses.

Moreover, we have observed previously in normotensive pregnant rats that, after crossing the placental barrier, SC is able to improve fetal and neonatal weights.11 Here, in hypertensive pregnant rats, we also obtained increased fetal weight at the end of pregnancy (day 18), which reached similar values to those of the control group soon after birth (PND10), and the placental weight of pre-eclamptic animals displayed the same response to SC. In addition, liver enlargement is a species-dependent effect of SC.26,41 Previous studies using the same PE model, but with higher SC doses, obtained similar values to those in the control group during pregnancy.19 Moreover, in humans, SC has been administered recently to mothers with early-onset severe fetal growth retardation to achieve increased fetal growth.15 Collectively, it seems that SC helps to improve fetal and placental weights, and even works for fetuses under hypoxic conditions.

Another important finding is the significantly lower offspring mortality rates in pre-eclamptic animals treated with SC with no long-term blood pressure effects in adult animals. SC enhances fetal tolerability to induced intrapartum asphyxia, and increases fetal weight in guinea pigs.42 Encouraging results are being obtained for the safety profile of SC in humans based on its use in neonates and pregnant women with pulmonary hypertension.43,44 There has only been one small study performed in sheep which showed adverse effects of SC following aggressive induced placental injury and after the administration of a very high dose of SC.45 The maternal plasma cell count is decreased in the l-NAME PE model, and this effect has also been corrected using SC.46

We chose to use the l-NAME animal model of PE because it has been validated in many studies. This model, like the others reported, causes only hypertension and proteinuria, but not the rest of the maternal syndrome seen in humans, although it also causes fetal growth restriction.17,47. The criticism of l-NAME is that it is a nonselective inhibitor of NOS,11,16,17,19,20,47 which includes possible tissue-specific differences in the expression of the NOS isoforms or in the availability of NOS to produce vascular relaxation.48 It could be argued that SC may only show benefits in the l-NAME model, but it has also been reported to be effective in the suramin animal model of PE.49

With regard to the haemodynamic results, and in contrast with humans, the uterine artery resistance index and PI in rats remain constant throughout gestation.11 Similar changes have also been reported in mice.50,51 This is probably influenced by the fact that rats have a larger number of embryos (average of 10–12) and much faster maternal and fetal heart rates (around 300 beats/minute). It is known that the faster the heart rate, the lower the ejection fraction and peak systolic velocity.52

In the group of rats with PE induced by l-NAME, one surprising finding is the decrease in the uterine artery PI on days E8 and E11 (Table 2). Apart from the above-mentioned haemodynamic differences between humans and rats, the interpretation of this concept is quite complex. It has also been reported that aortic stiffness increases and aortic systolic peak velocity decreases after the administration of l-NAME to pregnant rats.53 Similar changes have been observed in vivo in humans and animals with PE.54–56 These changes could affect the behaviour of uterine artery systolic peak velocity in rats with PE induced by l-NAME (Table 2). However, l-NAME has also been reported to induce arteriolar vasoconstriction in this animal PE model,48 which brings about an increase in pressure wave reflection and diastolic velocity. The aforementioned changes in systolic and diastolic velocities could partly explain the decrease in the uterine artery PI noted on day E8 in rats with PE induced by l-NAME.

When SC is administered to rats with induced PE, uterine haemodynamic behaviour returns to the pattern seen in control animals (Figure 3). On the fetal side, a decrease in the velocity of the DV a-wave in PE rats also returns to normal after SC administration (Figure 4A,B). As the DV a-wave represents the amount of blood reaching the heart from the placenta, this effect of SC is beneficial for the fetus. It is true that there are a number of aspects in this study that indicate the need for caution prior to any extrapolation to clinical practice, but understanding the mechanisms of action of SC may contribute to its effectiveness and safe use in complicated pregnancies in the near future.

In summary, our work demonstrates that the early administration of a low dose of SC, maintained throughout gestation, seems to be well tolerated by pre-eclamptic rats and is able to increase maternal uterine vascularisation and fetal supply, resulting in improved perinatal outcome. In future studies, it would be interesting to analyse whether SC administration can be limited only to the first trimester of pregnancy and whether or not these findings can be extrapolated to pregnant women.

Contribution to authorship

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

SH collected, analysed and interpreted the data, performed the statistical analysis and drafted the manuscript. BP conceived and designed the research, interpreted the data and drafted the manuscript. VS critically reviewed the manuscript for important intellectual content. OC contributed to data acquisition. JC conceived and designed the research, and critically reviewed the manuscript. VF conceived and designed the research, and managed funding and supervision. AP conceived and designed the research, and managed funding and supervision.

Details of ethics approval

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

The study was performed in accordance with European Directive 86/609/EEC and the National Institutes of Health (NIH) Guidelines for the welfare and use of laboratory animals. The study protocol used was approved by the Ethics Committee of the Centro de Investigación Príncipe Felipe (CIPF) in Valencia, Spain.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

This work was supported by a grant from the Fundación IVI-Instituto Universitario IVI, Universitat de Valencia, Valencia, Spain.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information
  • 1
    Duckitt K, Harrington D. Risk factors for pre-eclampsia at antenatal booking: systematic review of controlled studies. BMJ 2005;330:565.
  • 2
    Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet 2010;376:63144.
  • 3
    Hawkins TL, Roberts JM, Mangos GJ, Davis GK, Roberts LM, Brown MA. Plasma uric acid remains a marker of poor outcome in hypertensive pregnancy: a retrospective cohort study. BJOG 2012;119:48492.
  • 4
    Pijnenborg R, Vercruysse L, Verbist L, Van Assche FA. Interaction of interstitial trophoblast with placental bed capillaries and venules of normotensive and pre-eclamptic pregnancies. Placenta 1998;19:56975.
  • 5
    Merviel P, Carbillon L, Challier JC, Rabreau M, Beaufils M, Uzan S. Pathophysiology of preeclampsia: links with implantation disorders. Eur J Obstet Gynecol Reprod Biol 2004;115:13447.
  • 6
    Coppage KH, Sun X, Baker RS, Clark KE. Expression of phosphodiesterase 5 in maternal and fetal sheep. Am J Obstet Gynecol 2005;193:100510.
  • 7
    Maharaj CH, O’Toole D, Lynch T, Carney J, Jarman J, Higgins BD, et al. Effects and mechanisms of action of sildenafil citrate in human chorionic arteries. Reprod Biol Endocrinol 2009;7:34.
  • 8
    Zoma WDBR, Friedman A, Clark KE. Effects of combined use of sildenafil citrate (Viagra) and 17beta-estradiol on ovine coronary and uterine hemodynamics. Am J Obstet Gynecol 2004;190:12917.
  • 9
    Khan RN, Hamoud H, Warren A, Wong LF, Arulkumaran S. Relaxant action of sildenafil citrate (Viagra) on human myometrium of pregnancy. Am J Obstet Gynecol 2004;191:31521.
  • 10
    Mehats C, Schmitz T, Breuiller-Fouche M, Leroy MJ, Cabrol D. Should phosphodiesterase 5 selective inhibitors be used for uterine relaxation? Am J Obstet Gynecol 2006;195:1845.
  • 11
    Pellicer B, Herraiz S, Cauli O, Rodrigo R, Asensi M, Cortijo J, et al. Haemodynamic effects of long-term administration of sildenafil in normotensive pregnant and non-pregnant rats. BJOG 2011;118:61523.
  • 12
    Gori T, Sicuro S, Dragoni S, Donati G, Forconi S, Parker JD. Sildenafil prevents endothelial dysfunction induced by ischemia and reperfusion via opening of adenosine triphosphate-sensitive potassium channels: a human in vivo study. Circulation 2005;111:7426.
  • 13
    Wareing M, Myers JE, O’Hara M, Baker PN. Sildenafil citrate (Viagra) enhances vasodilatation in fetal growth restriction. J Clin Endocrinol Metab 2005;90:25505.
  • 14
    Wareing M, Myers JE, O’Hara M, Kenny LC, Taggart MJ, Skillern L, et al. Phosphodiesterase-5 inhibitors and omental and placental small artery function in normal pregnancy and pre-eclampsia. Eur J Obstet Gynecol Reprod Biol 2006;127:419.
  • 15
    Von Dadelszen P, Dwinnell S, Magee LA, Carleton BC, Gruslin A, Lee B, et al. Sildenafil citrate therapy for severe early-onset intrauterine growth restriction. BJOG 2011;118:6248.
  • 16
    Cauli O, Herraiz S, Pellicer B, Pellicer A, Felipo V. Treatment with sildenafil prevents impairment of learning in rats born to pre-eclamptic mothers. Neuroscience 2010;171:50612.
  • 17
    Fernandez Celadilla L, Carbajo Rueda M, Munoz Rodriguez M. Prolonged inhibition of nitric oxide synthesis in pregnant rats: effects on blood pressure, fetal growth and litter size. Arch Gynecol Obstet 2005;271:2438.
  • 18
    Pellicer B, Herraiz S, Leal A, Simon C, Pellicer A. Prenatal brain damage in preeclamptic animal model induced by gestational nitric oxide synthase inhibition. J Pregnancy 2011;2011:809569.
  • 19
    Ramesar SV, Mackraj I, Gathiram P, Moodley J. Sildenafil citrate improves fetal outcomes in pregnant, L-NAME treated, Sprague-Dawley rats. Eur J Obstet Gynecol Reprod Biol 2010;149:226.
  • 20
    Ramesar SV, Mackraj I, Gathiram P, Moodley J. Sildenafil citrate decreases sFlt-1 and sEng in pregnant l-NAME treated Sprague-Dawley rats. Eur J Obstet Gynecol Reprod Biol 2011;157:13640.
  • 21
    Baquero H, Soliz A, Neira F, Venegas ME, Sola A. Oral sildenafil in infants with persistent pulmonary hypertension of the newborn: a pilot randomized blinded study. Pediatrics 2006;117:107783.
  • 22
    Dussault S, Maingrette F, Menard C, Michaud SE, Haddad P, Groleau J, et al. Sildenafil increases endothelial progenitor cell function and improves ischemia-induced neovascularization in hypercholesterolemic apolipoprotein E-deficient mice. Hypertension 2009;54:10439.
  • 23
    Erceg S, Monfort P, Hernandez-Viadel M, Rodrigo R, Montoliu C, Felipo V. Oral administration of sildenafil restores learning ability in rats with hyperammonemia and with portacaval shunts. Hepatology 2005;41:299306.
  • 24
    Gomez-Pinedo U, Rodrigo R, Cauli O, Herraiz S, Garcia-Verdugo JM, Pellicer B, et al. cGMP modulates stem cell differentiation to neurons in brain in vivo. Neuroscience 2010;165:127583.
  • 25
    Haase E, Bigam DL, Cravetchi O, Cheung PY. Dose response of intravenous sildenafil on systemic and regional hemodynamics in hypoxic neonatal piglets. Shock 2006;26:99106.
  • 26
    Villanueva-Garcia D, Mota-Rojas D, Hernandez-Gonzalez R, Sanchez-Aparicio P, Alonso-Spilsbury M, Trujillo-Ortega ME, et al. A systematic review of experimental and clinical studies of sildenafil citrate for intrauterine growth restriction and pre-term labour. J Obstet Gynaecol 2007;27:2559.
  • 27
    Felfernig-Boehm D, Salat A, Vogl SE, Murabito M, Felfernig M, Schmidt D, et al. Early detection of preeclampsia by determination of platelet aggregability. Thromb Res 2000;98:13946.
  • 28
    Rodriguez W, Mold C, Kataranovski M, Hutt J, Marnell LL, Du Clos TW. Reversal of ongoing proteinuria in autoimmune mice by treatment with C-reactive protein. Arthr Rheum 2005;52:64250.
  • 29
    Zenclussen AC. A novel mouse model for preeclampsia by transferring activated th1 cells into normal pregnant mice. Methods Molec Med 2006;122:40112.
  • 30
    Caluwaerts S, Vercruysse L, Luyten C, Pijnenborg R. Endovascular trophoblast invasion and associated structural changes in uterine spiral arteries of the pregnant rat. Placenta 2005;26:57484.
  • 31
    De Rijk EPVEE, Flik G. Pregnancy dating in the rat: placental morphology and maternal blood parameters. Toxicol Pathol 2002;30:27182.
  • 32
    Agarwal D, Haque M, Sriramula S, Mariappan N, Pariaut R, Francis J. Role of proinflammatory cytokines and redox homeostasis in exercise-induced delayed progression of hypertension in spontaneously hypertensive rats. Hypertension 2009;54:1393400.
  • 33
    Thida M, Earl J, Zhao Y, Wang H, Tse CS, Vickers JJ, et al. Effects of sepiapterin supplementation and NOS inhibition on glucocorticoid-induced hypertension. Am J Hypertens 2010;23:56974.
  • 34
    Yzydorczyk C, Comte B, Cambonie G, Lavoie JC, Germain N, Ting Shun Y, et al. Neonatal oxygen exposure in rats leads to cardiovascular and renal alterations in adulthood. Hypertension 2008;52:88995.
  • 35
    Pellicer B, Herraiz S, Taboas E, Felipo V, Simon C, Pellicer A. Ultrasound bioeffects in rats: quantification of cellular damage in the fetal liver after pulsed Doppler imaging. Ultrasound Obstet Gynecol 2011;37:6438.
  • 36
    Hecher K, Campbell S, Snijders R, Nicolaides K. Reference ranges for fetal venous and atrioventricular blood flow parameters. Ultrasound Obstet Gynecol 1994;4:38190.
  • 37
    Hecher K, Snijders R, Campbell S, Nicolaides K. Fetal venous, intracardiac, and arterial blood flow measurements in intrauterine growth retardation: relationship with fetal blood gases. Am J Obstet Gynecol 1995;173:105.
  • 38
    Vidavalur R, Penumathsa SV, Thirunavukkarasu M, Zhan L, Krueger W, Maulik N. Sildenafil augments early protective transcriptional changes after ischemia in mouse myocardium. Gene 2009;2:307.
  • 39
    Vidavalur R, Penumathsa SV, Zhan L, Thirunavukkarasu M, Maulik N. Sildenafil induces angiogenic response in human coronary arteriolar endothelial cells through the expression of thioredoxin, hemeoxygenase and vascular endothelial growth factor. Vascul Pharmacol 2006;45:915.
  • 40
    Vural P, Degirmencioglu S, Saral NY, Demirkan A, Akgul C, Yildirim G, et al. Tumor necrosis factor alpha, interleukin-6 and interleukin-10 polymorphisms in preeclampsia. J Obstet Gynaecol Res 2010;36:6471.
  • 41
    Abbott D, Comby P, Charuel C, Graepel P, Hanton G, Leblanc B, et al. Preclinical safety profile of sildenafil. Int J Impotence Res 2004;16:498504.
  • 42
    Sanchez-Aparicio P, Mota-Rojas D, Nava-Ocampo AA, Trujillo-Ortega ME, Alfaro-Rodriguez A, Arch E, et al. Effects of sildenafil on the fetal growth of guinea pigs and their ability to survive induced intrapartum asphyxia. Am J Obstet Gynecol 2008;198:127e16.
  • 43
    Huang S, DeSantis ER. Treatment of pulmonary arterial hypertension in pregnancy. Am J Health Syst Pharm 2007;64:19226.
  • 44
    Humpl T, Reyes JT, Erickson S, Armano R, Holtby H, Adatia I. Sildenafil therapy for neonatal and childhood pulmonary hypertensive vascular disease. Cardiol Young 2011;21:18793.
  • 45
    Miller SL, Loose JM, Jenkin G, Wallace EM. The effects of sildenafil citrate (Viagra) on uterine blood flow and well being in the intrauterine growth-restricted fetus. Am J Obstet Gynecol 2009;200:102e17.
  • 46
    Ravishankar V, Buhimschi CS, Booth CJ, Bhandari V, Norwitz E, Copel J, et al. Fetal nucleated red blood cells in a rat model of intrauterine growth restriction induced by hypoxia and nitric oxide synthase inhibition. Am J Obstet Gynecol 2007;196:482e18.
  • 47
    Carbajo Rueda M, Munoz Rodriguez M, Fernandez Celadilla L. IGF-I, 17beta-estradiol and progesterone in SHR and in rats treated with L-NAME: fetal–placental development. Arch Gynecol Obstet 2004;270:2359.
  • 48
    Khalil RA, Granger JP. Vascular mechanisms of increased arterial pressure in preeclampsia: lessons from animal models. Am J Physiol Regul Integr Comp Physiol 2002;283:R2945.
  • 49
    Turgut NH, Temiz TK, Bagcivan I, Turgut B, Gulturk S, Karadas B. The effect of sildenafil on the altered thoracic aorta smooth muscle responses in rat pre-eclampsia model. Eur J Pharmacol 2008;3:1807.
  • 50
    Geusens N, Verlohren S, Luyten C, Taube M, Hering L, Vercruysse L, et al. Endovascular trophoblast invasion, spiral artery remodelling and uteroplacental haemodynamics in a transgenic rat model of pre-eclampsia. Placenta 2008;29:61423.
  • 51
    Verlohren S, Niehoff M, Hering L, Geusens N, Herse F, Tintu AN, et al. Uterine vascular function in a transgenic preeclampsia rat model. Hypertension 2008;51:54753.
  • 52
    Maulik D. Spectral Doppler Sonography: Waveforms Analysis and Hemodynamic Interpretation. Berlin, Heidelberg: Springer-Verlag, 2005.
  • 53
    Fitch RM, Vergona R, Sullivan ME, Wang YX. Nitric oxide synthase inhibition increases aortic stiffness measured by pulse wave velocity in rats. Cardiovasc Res 2001;51:3518.
  • 54
    Elvan-Taspinar A, Franx A, Bots ML, Koomans HA, Bruinse HW. Arterial stiffness and fetal growth in normotensive pregnancy. Am J Hypertens 2005;18:33741.
  • 55
    Ronnback M, Lampinen K, Groop PH, Kaaja R. Pulse wave reflection in currently and previously preeclamptic women. Hypertens Pregnancy 2005;24:17180.
  • 56
    Spasojevic M, Smith SA, Morris JM, Gallery ED. Peripheral arterial pulse wave analysis in women with pre-eclampsia and gestational hypertension. BJOG 2005;112:14758.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interests
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
  13. Supporting Information

Figure S1. Doppler flow velocity waveforms from the uterine artery in pregnant rats.

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BJO_3430_sm_FigS1.pdf288KSupporting info item

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