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EURO PEAN SO CIETY O F CARDIOLOGY ®
Original scientific paper
Antioxidants improve vascular function in children conceived by assisted reproductive technologies: A randomized double-blind placebo-controlled trial
European Journal of Preventive Cardiology 0(00) 1–9 ! The European Society of Cardiology 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/2047487314535117 ejpc.sagepub.com
Stefano F Rimoldi1,*, Claudio Sartori1,2,*, Emrush Rexhaj1, Damian M Bailey3, Stefano F de Marchi1, Jane McEneny4, Robert von Arx1, David Cerny1, Herve´ Duplain5, Marc Germond6, Yves Allemann1,* and Urs Scherrer1,7,*
Abstract Aims: Children conceived by assisted reproductive technology (ART) display vascular dysfunction. Its underlying mechanism, potential reversibility and long-term consequences for cardiovascular risk are unknown. In mice, ART induces arterial hypertension and shortens the life span. These problems are related to decreased vascular endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis. The aim of this study was to determine whether ART-induced vascular dysfunction in humans is related to a similar mechanism and potentially reversible. To this end we tested whether antioxidants improve endothelial function by scavenging free radicals and increasing NO bioavailability. Methods and results: In this prospective double-blind placebo controlled study in 21 ART and 21 control children we assessed the effects of a four-week oral supplementation with antioxidant vitamins C (1 g) and E (400 IU) or placebo (allocation ratio 2:1) on flow-mediated vasodilation (FMD) of the brachial artery and pulmonary artery pressure (echocardiography) during high-altitude exposure (3454 m), a manoeuver known to facilitate the detection of pulmonary vascular dysfunction and to decrease NO bioavailability by stimulating oxidative stress. Antioxidant supplementation significantly increased plasma NO measured by ozone-based chemiluminescence (from 21.7 7.9 to 26.9 7.6 mM, p ¼ 0.04) and FMD (from 7.0 2.1 to 8.7 2.0%, p ¼ 0.004) and attenuated altitude-induced pulmonary hypertension (from 33 8 to 28 6 mm Hg, p ¼ 0.028) in ART children, whereas it had no detectable effect in control children. Conclusions: Antioxidant administration to ART children improved NO bioavailability and vascular responsiveness in the systemic and pulmonary circulation. Collectively, these findings indicate that in young individuals ART-induced vascular dysfunction is subject to redox regulation and reversible. Keywords Endothelial dysfunction, vitamin, nitric oxide, in vitro fertilization Received 18 March 2014; accepted 19 April 2014
Introduction Studies in experimental animals and humans demonstrate that adverse events during early life cause alterations of cardiovascular function that may increase cardiovascular risk later in life.1 The recent observation of generalized vascular dysfunction in children conceived by assisted reproductive technology (ART) provides another example of this problem, but the underlying mechanism is unknown.2,3 To provide insight into underpinning mechanisms we studied
1
Department of Cardiology and Clinical Research, Inselspital, University Hospital, Bern, Switzerland 2 Department of Internal Medicine, Centre Hospitalieruniversitaire Vaudois (CHUV), Lausanne, Switzerland 3 Neurovascular Research Laboratory, University of South Wales, UK 4 Centre for Clinical and Population Sciences, Queen’s University, Northern Ireland 5 Department of Internal Medicine, Dele´mont, Switzerland 6 Centre de Procre´ation Me´dicalement Assiste´e, Lausanne, Switzerland 7 Facultad de Ciencias, Departamento de Biologı´a, Universidad de Tarapaca´, Chile *These authors contributed equally to this work. Corresponding author: Stefano F Rimoldi, Department of Cardiology and Clinical Research, Inselspital, University Hospital, Bern 3010, Switzerland. Email: [email protected]
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ART mice. We found that ART mice display endothelial dysfunction and arterial hypertension4 which are related to decreased vascular endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis caused by epigenetic alterations of the eNOS gene.4 We speculated that a similar mechanism contributes to vascular dysfunction in ART children. In humans with endothelial dysfunction, antioxidant vitamins improve endothelial function by scavenging free radicals and increasing NO bioavailability.5,6 To test this hypothesis, we performed a prospective double-blind placebo controlled study in ART and control children to assess the effects of a four-week antioxidant supplementation with vitamins C and E on systemic and pulmonary vascular function during high-altitude exposure (3454 m), a manoeuver known to facilitate the detection of pulmonary vascular dysfunction and to decrease NO bioavailability by stimulating oxidative stress.7–9
Methods Study design This was a single-center, randomized, double-blind, placebo-controlled trial in ART and age- and gendermatched healthy control children. The participants were exposed to high altitude (3554 m) during 48 h, the first time without any treatment, the second time
after four weeks with oral supplementation with antioxidant vitamins or placebo (Figure 1). The allocation ratio was 2:1 for treatment with antioxidant vitamins or placebo.
Participants We recruited 21 children conceived by ART and 21 age-and gender-matched healthy controls. Children conceived by ART and control children who had participated in a previous high-altitude study3 were contacted by letter, and those who agreed to participate and met the inclusion criteria were recruited. Inclusion criteria were: singletons, born at term (>37 weeks gestational age) with normal birth weight (>2500 g) and without any gestational or perinatal complications. Exclusion criteria included any acute illness or medical treatment, high altitude (>2500 m) exposure during the last three months preceding the study. None of the participants was taking any antioxidant/anti-inflammatory preparations/supplements known to influence the redox state.
Setting and data collection All measurements were performed at high altitude (Jungfraujoch, 3554 m, Switzerland). The children ascended to the high-altitude research station by train
Control children (n=21)
ART children (n=21)
First high-altitude exposure (assessment of systemic and pulmonary vascular function)
Randomization
ART + Vitamin children (n=13)
ART + Placebo children (n=8)
Control + Vitamin children (n=13)
Control + Placebo children (n=8)
4 weeks vitamin C + E (1g + 400 IU / day) or placebo
Second high-altitude exposure (assessment of systemic and pulmonary vascular function and oxidative stress)
Figure 1. Flow chart of the study design. ART: assisted reproductive technology.
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and spent two days and two nights at this laboratory. Measurements were performed on the morning before descent.
Interventions Five weeks before the second high-altitude exposure, each participant received two packs, one labeled vit C/ placebo (60 pills, one pill containing 500 mg ascorbic acid, Burgerstein Vitamin C retard, ref. 08164/09 or corresponding placebo) the other labeled vit E/placebo (30 pills, one pill containing 400 IU a-tocopherol, Burgerstein Vitamin E, ref. 263226 or corresponding placebo), with the personal code, the name and surname of the participant and the starting and finishing date of the treatment. Beginning on the date indicated on the packs, for 28 days each participant took two pills every morning from the pack labeled vit C/placebo and one pill from the pack labeled vit E/placebo. The total daily dose was 1 g vitamin C and 400 IU vitamin E. To assess adherence, each participant completed a daily questionnaire and the number of remaining pills was counted at the end of the treatment. Participants were encouraged to follow a low nitrate/nitrite diet prior to and throughout the duration of the study with specific instructions to avoid fruits, salads and cured meats.10
Procedure The protocol was approved by the institutional review board on human investigation of the University of Lausanne (Protocol # 17/09), Switzerland, and was registered (www.clinicaltrials.gov; registration No. NCT00837642). All parents provided written informed consent. Randomization was performed via a computer-based system generating an allocation sequence in randomly varying blocks. All participants and investigators were blinded.
Main outcome and measures Primary outcomes were: changes in systemic vascular function assessed by flow-mediated vasodilation (FMD) of the brachial artery and carotid-femoral pulse wave velocity (PWV), and changes in pulmonary vascular function assessed by estimation of pulmonary artery pressure using Doppler echocardiography. The secondary end point was the intra-individual change in the plasma concentration of nitric oxide.
Assessment of systemic vascular function Systemic vascular function was examined after 15 min of rest in the supine position in a temperature-controlled room (22 C) using gold standard methods to
non-invasively assess endothelial function and arterial stiffness.11–13 Systemic conduit artery endothelial function was assessed by measuring the increase of the brachial artery diameter evoked by reactive hyperemia induced by a pressure cuff placed around the distal forearm with a high-resolution ultrasound probe (Acuson Sequoia C 512; Acuson Siemens, Mountain View, California, USA) and using an automatic wall tracking software (FMD Studio, Computer Vision Group, Pisa, Italy) according to international guidelines14,15 and as previously described.3,16–18 The coefficient of variation between two measurements in the same 30 subjects 24 h apart was 5.2%.3 FMD was expressed as the maximal percentage change in vessel diameter from baseline. Large artery stiffness was assessed non-invasively by measuring carotid-femoral PWV with the Complior device (Artech Medical, Pantin, France) according to international guidelines as described previously.3,18 The coefficient of variation between two measurements in the same 30 subjects 24 h apart was 6.3%.3
Assessment of pulmonary vascular function To estimate systolic pulmonary artery pressure and cardiac output, echocardiographic recordings were obtained with a real-time, phased-array sector scanner (Acuson Sequoia C 512; Acuson Siemens, Mountain View, California, USA) as previously described.3,17,19,20 Briefly, after tricuspid regurgitation had been localized with Doppler color-flow imagining, the pressure gradient between the right ventricle (RV) and the right atrium (RA) was calculated using the modified Bernoulli equation. In children at this altitude, the intra- and inter-observer variability of the RV-RA pressure gradient measurements (n ¼ 30) was 5.1 and 6.0% respectively.20 Cardiac output was assessed as previously described20 and cardiac index was calculated by dividing cardiac output (l/min) by the body surface area (m2).
Arterial oxygen saturation and heart rate Transcutaneous arterial oxygen saturation and heart rate were measured at a fingertip with a pulse oxymeter (OxiMax N-65, Nellcor, Pleasanton, California, USA).
Metabolic assessment All subjects provided a 12-hour overnight-fasting, resting, blood sample from a catheter located in a forearm antecubital vein. Samples were immediately centrifuged at 600 g (4 C) for 10 min; the supernatant (K-EDTA plasma) was snap-frozen without delay and stored under liquid nitrogen prior to transport
Downloaded from cpr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 29, 2015
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(Cryopak CP100; Taylor-Wharton International) for batch analysis. Nitric oxide (NO). Ozone-based chemiluminescence (OBC Model 280i; NOA, Sievers, Boulder, Colorado, USA), arguably the most sensitive technique for the molecular detection of NO in human blood was employed for the detection of plasma and red blood cell (RBC) bound NO metabolites. Samples (20 ml) were analyzed for the total concentration of plasma NO (nitrate þ nitrite þ Snitrosothiols) by vanadium (III) reduction.21 All calculations were performed using Origin/Peak Analysis software. The intra- and inter-assay coefficients of variation were 7% and 10%, respectively. Ascorbate radical. Direct detection of the long-lived ascorbate radical (A–) was employed as a measure of ‘‘global’’ free radical formation using X-band (9.79 GHz) electron paramagnetic resonance spectroscopy (Bruker, Karlsruhe, Germany) as previously described.16 K-EDTA plasma (1 ml) was injected into a high-sensitivity multiple-bore sample cell (AquaX, Bruker Instruments Inc., Billerica, USA) and the characteristic doublet of A– (aH ¼ 1.76 G) was recorded 12 min after the end of plasma recovery by signal averaging three scans with the following parameters: resolution, 1024 points: microwave power, 20 mW; modulation amplitude, 0.65 Gauss (G); receiver gain, 2 105; time constant, 40.96 ms; sweep rate, 0.25 G/s and sweep width, 15 G. All spectra were filtered identically using Bruker WinEPR 2.11 software and the double integral calculated using Origin software. The intra- and inter-assay coefficients of variation were both
(CPR)
[1–9] [PREPRINTER stage]
EURO PEAN SO CIETY O F CARDIOLOGY ®
Original scientific paper
Antioxidants improve vascular function in children conceived by assisted reproductive technologies: A randomized double-blind placebo-controlled trial
European Journal of Preventive Cardiology 0(00) 1–9 ! The European Society of Cardiology 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/2047487314535117 ejpc.sagepub.com
Stefano F Rimoldi1,*, Claudio Sartori1,2,*, Emrush Rexhaj1, Damian M Bailey3, Stefano F de Marchi1, Jane McEneny4, Robert von Arx1, David Cerny1, Herve´ Duplain5, Marc Germond6, Yves Allemann1,* and Urs Scherrer1,7,*
Abstract Aims: Children conceived by assisted reproductive technology (ART) display vascular dysfunction. Its underlying mechanism, potential reversibility and long-term consequences for cardiovascular risk are unknown. In mice, ART induces arterial hypertension and shortens the life span. These problems are related to decreased vascular endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis. The aim of this study was to determine whether ART-induced vascular dysfunction in humans is related to a similar mechanism and potentially reversible. To this end we tested whether antioxidants improve endothelial function by scavenging free radicals and increasing NO bioavailability. Methods and results: In this prospective double-blind placebo controlled study in 21 ART and 21 control children we assessed the effects of a four-week oral supplementation with antioxidant vitamins C (1 g) and E (400 IU) or placebo (allocation ratio 2:1) on flow-mediated vasodilation (FMD) of the brachial artery and pulmonary artery pressure (echocardiography) during high-altitude exposure (3454 m), a manoeuver known to facilitate the detection of pulmonary vascular dysfunction and to decrease NO bioavailability by stimulating oxidative stress. Antioxidant supplementation significantly increased plasma NO measured by ozone-based chemiluminescence (from 21.7 7.9 to 26.9 7.6 mM, p ¼ 0.04) and FMD (from 7.0 2.1 to 8.7 2.0%, p ¼ 0.004) and attenuated altitude-induced pulmonary hypertension (from 33 8 to 28 6 mm Hg, p ¼ 0.028) in ART children, whereas it had no detectable effect in control children. Conclusions: Antioxidant administration to ART children improved NO bioavailability and vascular responsiveness in the systemic and pulmonary circulation. Collectively, these findings indicate that in young individuals ART-induced vascular dysfunction is subject to redox regulation and reversible. Keywords Endothelial dysfunction, vitamin, nitric oxide, in vitro fertilization Received 18 March 2014; accepted 19 April 2014
Introduction Studies in experimental animals and humans demonstrate that adverse events during early life cause alterations of cardiovascular function that may increase cardiovascular risk later in life.1 The recent observation of generalized vascular dysfunction in children conceived by assisted reproductive technology (ART) provides another example of this problem, but the underlying mechanism is unknown.2,3 To provide insight into underpinning mechanisms we studied
1
Department of Cardiology and Clinical Research, Inselspital, University Hospital, Bern, Switzerland 2 Department of Internal Medicine, Centre Hospitalieruniversitaire Vaudois (CHUV), Lausanne, Switzerland 3 Neurovascular Research Laboratory, University of South Wales, UK 4 Centre for Clinical and Population Sciences, Queen’s University, Northern Ireland 5 Department of Internal Medicine, Dele´mont, Switzerland 6 Centre de Procre´ation Me´dicalement Assiste´e, Lausanne, Switzerland 7 Facultad de Ciencias, Departamento de Biologı´a, Universidad de Tarapaca´, Chile *These authors contributed equally to this work. Corresponding author: Stefano F Rimoldi, Department of Cardiology and Clinical Research, Inselspital, University Hospital, Bern 3010, Switzerland. Email: [email protected]
Downloaded from cpr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 29, 2015
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ART mice. We found that ART mice display endothelial dysfunction and arterial hypertension4 which are related to decreased vascular endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis caused by epigenetic alterations of the eNOS gene.4 We speculated that a similar mechanism contributes to vascular dysfunction in ART children. In humans with endothelial dysfunction, antioxidant vitamins improve endothelial function by scavenging free radicals and increasing NO bioavailability.5,6 To test this hypothesis, we performed a prospective double-blind placebo controlled study in ART and control children to assess the effects of a four-week antioxidant supplementation with vitamins C and E on systemic and pulmonary vascular function during high-altitude exposure (3454 m), a manoeuver known to facilitate the detection of pulmonary vascular dysfunction and to decrease NO bioavailability by stimulating oxidative stress.7–9
Methods Study design This was a single-center, randomized, double-blind, placebo-controlled trial in ART and age- and gendermatched healthy control children. The participants were exposed to high altitude (3554 m) during 48 h, the first time without any treatment, the second time
after four weeks with oral supplementation with antioxidant vitamins or placebo (Figure 1). The allocation ratio was 2:1 for treatment with antioxidant vitamins or placebo.
Participants We recruited 21 children conceived by ART and 21 age-and gender-matched healthy controls. Children conceived by ART and control children who had participated in a previous high-altitude study3 were contacted by letter, and those who agreed to participate and met the inclusion criteria were recruited. Inclusion criteria were: singletons, born at term (>37 weeks gestational age) with normal birth weight (>2500 g) and without any gestational or perinatal complications. Exclusion criteria included any acute illness or medical treatment, high altitude (>2500 m) exposure during the last three months preceding the study. None of the participants was taking any antioxidant/anti-inflammatory preparations/supplements known to influence the redox state.
Setting and data collection All measurements were performed at high altitude (Jungfraujoch, 3554 m, Switzerland). The children ascended to the high-altitude research station by train
Control children (n=21)
ART children (n=21)
First high-altitude exposure (assessment of systemic and pulmonary vascular function)
Randomization
ART + Vitamin children (n=13)
ART + Placebo children (n=8)
Control + Vitamin children (n=13)
Control + Placebo children (n=8)
4 weeks vitamin C + E (1g + 400 IU / day) or placebo
Second high-altitude exposure (assessment of systemic and pulmonary vascular function and oxidative stress)
Figure 1. Flow chart of the study design. ART: assisted reproductive technology.
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and spent two days and two nights at this laboratory. Measurements were performed on the morning before descent.
Interventions Five weeks before the second high-altitude exposure, each participant received two packs, one labeled vit C/ placebo (60 pills, one pill containing 500 mg ascorbic acid, Burgerstein Vitamin C retard, ref. 08164/09 or corresponding placebo) the other labeled vit E/placebo (30 pills, one pill containing 400 IU a-tocopherol, Burgerstein Vitamin E, ref. 263226 or corresponding placebo), with the personal code, the name and surname of the participant and the starting and finishing date of the treatment. Beginning on the date indicated on the packs, for 28 days each participant took two pills every morning from the pack labeled vit C/placebo and one pill from the pack labeled vit E/placebo. The total daily dose was 1 g vitamin C and 400 IU vitamin E. To assess adherence, each participant completed a daily questionnaire and the number of remaining pills was counted at the end of the treatment. Participants were encouraged to follow a low nitrate/nitrite diet prior to and throughout the duration of the study with specific instructions to avoid fruits, salads and cured meats.10
Procedure The protocol was approved by the institutional review board on human investigation of the University of Lausanne (Protocol # 17/09), Switzerland, and was registered (www.clinicaltrials.gov; registration No. NCT00837642). All parents provided written informed consent. Randomization was performed via a computer-based system generating an allocation sequence in randomly varying blocks. All participants and investigators were blinded.
Main outcome and measures Primary outcomes were: changes in systemic vascular function assessed by flow-mediated vasodilation (FMD) of the brachial artery and carotid-femoral pulse wave velocity (PWV), and changes in pulmonary vascular function assessed by estimation of pulmonary artery pressure using Doppler echocardiography. The secondary end point was the intra-individual change in the plasma concentration of nitric oxide.
Assessment of systemic vascular function Systemic vascular function was examined after 15 min of rest in the supine position in a temperature-controlled room (22 C) using gold standard methods to
non-invasively assess endothelial function and arterial stiffness.11–13 Systemic conduit artery endothelial function was assessed by measuring the increase of the brachial artery diameter evoked by reactive hyperemia induced by a pressure cuff placed around the distal forearm with a high-resolution ultrasound probe (Acuson Sequoia C 512; Acuson Siemens, Mountain View, California, USA) and using an automatic wall tracking software (FMD Studio, Computer Vision Group, Pisa, Italy) according to international guidelines14,15 and as previously described.3,16–18 The coefficient of variation between two measurements in the same 30 subjects 24 h apart was 5.2%.3 FMD was expressed as the maximal percentage change in vessel diameter from baseline. Large artery stiffness was assessed non-invasively by measuring carotid-femoral PWV with the Complior device (Artech Medical, Pantin, France) according to international guidelines as described previously.3,18 The coefficient of variation between two measurements in the same 30 subjects 24 h apart was 6.3%.3
Assessment of pulmonary vascular function To estimate systolic pulmonary artery pressure and cardiac output, echocardiographic recordings were obtained with a real-time, phased-array sector scanner (Acuson Sequoia C 512; Acuson Siemens, Mountain View, California, USA) as previously described.3,17,19,20 Briefly, after tricuspid regurgitation had been localized with Doppler color-flow imagining, the pressure gradient between the right ventricle (RV) and the right atrium (RA) was calculated using the modified Bernoulli equation. In children at this altitude, the intra- and inter-observer variability of the RV-RA pressure gradient measurements (n ¼ 30) was 5.1 and 6.0% respectively.20 Cardiac output was assessed as previously described20 and cardiac index was calculated by dividing cardiac output (l/min) by the body surface area (m2).
Arterial oxygen saturation and heart rate Transcutaneous arterial oxygen saturation and heart rate were measured at a fingertip with a pulse oxymeter (OxiMax N-65, Nellcor, Pleasanton, California, USA).
Metabolic assessment All subjects provided a 12-hour overnight-fasting, resting, blood sample from a catheter located in a forearm antecubital vein. Samples were immediately centrifuged at 600 g (4 C) for 10 min; the supernatant (K-EDTA plasma) was snap-frozen without delay and stored under liquid nitrogen prior to transport
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(Cryopak CP100; Taylor-Wharton International) for batch analysis. Nitric oxide (NO). Ozone-based chemiluminescence (OBC Model 280i; NOA, Sievers, Boulder, Colorado, USA), arguably the most sensitive technique for the molecular detection of NO in human blood was employed for the detection of plasma and red blood cell (RBC) bound NO metabolites. Samples (20 ml) were analyzed for the total concentration of plasma NO (nitrate þ nitrite þ Snitrosothiols) by vanadium (III) reduction.21 All calculations were performed using Origin/Peak Analysis software. The intra- and inter-assay coefficients of variation were 7% and 10%, respectively. Ascorbate radical. Direct detection of the long-lived ascorbate radical (A–) was employed as a measure of ‘‘global’’ free radical formation using X-band (9.79 GHz) electron paramagnetic resonance spectroscopy (Bruker, Karlsruhe, Germany) as previously described.16 K-EDTA plasma (1 ml) was injected into a high-sensitivity multiple-bore sample cell (AquaX, Bruker Instruments Inc., Billerica, USA) and the characteristic doublet of A– (aH ¼ 1.76 G) was recorded 12 min after the end of plasma recovery by signal averaging three scans with the following parameters: resolution, 1024 points: microwave power, 20 mW; modulation amplitude, 0.65 Gauss (G); receiver gain, 2 105; time constant, 40.96 ms; sweep rate, 0.25 G/s and sweep width, 15 G. All spectra were filtered identically using Bruker WinEPR 2.11 software and the double integral calculated using Origin software. The intra- and inter-assay coefficients of variation were both
