This study was undertaken to compare the concentrations of pro- and anti-angiogenic growth factors, nitric oxide (NO) stable metabolites in maternal serum and embryonic left ventricular (LV) isovolumic relaxation time (IRT, ms) during the first trimester in two groups of women: with pregnancy conceived by assisted reproductive technologies (ART, nј39) and normally conceived (control group, nј68) pregnancy. The concentration of vasoconstrictor endothelin 1 was 45.5 times more in ART than in control group. On the contrary, the concentrations of NO stable metabolites in ART were 1.9 times less than in control women. The assessment of angiogenic suppressors in ART women demonstrates the decrease in s-endoglin concentration was 1.6 times and in soluble receptor to vascular endothelial growth factor concentration was 2.0 times in comparison with control group. There was a significant increase in LV IRT in ART embryos in comparison to control ones. These data suggest significant changes in pro-antiangiogenic factors balance and increase in vascular impedance in ART-conceived embryos

Bashmakova, N. V.

Chistiakova, G. N.

Gazieva, I. A.

Mazurov, D. O.

Trapeznikova, Y. M.

Tsyvian, P. B.

Ural State Medical University Repository

http://informahealthcare.com/gyeISSN: 0951-3590 (print), 1473-0766 (electronic)Gynecol Endocrinol, Early Online: 1–3! 2015 Mother and Child Care Research Institute. DOI: 10.3109/09513590.2015.1085199ORIGINAL ARTICLEEndothelial function, regulation of angiogenesis and embryonic centralhemodynamics in ART-conceived pregnanciesN. V. Bashmakova1, P. B. Tsyvian1,2,3, G. N. Chistiakova1, I. A. Gazieva1, Y. M. Trapeznikova1, and D. O. Mazurov11Mother and Child Care Research Institute, Russian Ministry of Public Health, Yekaterinburg, Russia, 2Ural State Medical University, Yekaterinburg,Russia, and 3Institute of Immunology and Physiology, Ural Branch of Russian Academy of Sciences, Yekaterinburg, RussiaQ1AbstractThis study was undertaken to compare the concentrations of pro- and anti-angiogenic growthfactors, nitric oxide (NO) stable metabolites in maternal serum and embryonic left ventricular(LV) isovolumic relaxation time (IRT, ms) during the first trimester in two groups of women: withpregnancy conceived by assisted reproductive technologies (ART, n¼ 39) and normallyconceived (control group, n¼ 68) pregnancy. The concentration of vasoconstrictor endothelin 1was 45.5 times more in ART than in control group. On the contrary, the concentrations of NOstable metabolites in ART were 1.9 times less than in control women. The assessment ofangiogenic suppressors in ART women demonstrates the decrease in s-endoglin concentrationwas 1.6 times and in soluble receptor to vascular endothelial growth factor concentration was2.0 times in comparison with control group. There was a significant increase in LV IRT in ARTembryos in comparison to control ones. These data suggest significant changes in pro- anti-angiogenic factors balance and increase in vascular impedance in ART-conceived embryos.KeywordsAssisted reproductive technologies,embryonic vascular impedance,pro- anti-angiogenic factors balanceHistoryReceived 2 2 2Revised 2 2 2Accepted 2 2 2Published online 2 2 2IntroductionAssisted reproductive technologies (ART) have aided millions ofcouples worldwide to have children. In vitro fertilization has beenperformed for more than three decades, and children born after ARTnow estimated up 1–4% of the births in developed countries [1]. Withcontinued ART success and utilization, any long-term health risks dueto ART treatment have the potential to affect a substantial proportionof the population and increase the future health care burden.Epidemiological work in humans has put forward the hypoth-esis that intrauterine environmental influences may predispose thechildren to chronic cardiovascular and metabolic disease inadulthood [2]. Therefore, the safety of ART for long-term healthhas a growing importance, but there is little information on thisissue. This could be related, at least in part, to the young age ofthese persons because clinically manifest disease may not yet havehad time to develop.Although ART are generally considered safe, the potentialassociation of these technologies with poorer pregnancy outcomeshas long been investigated. There is evidence that ART isassociated with increased risk for adverse perinatal outcome andcongenital malformations [3]. Preliminary evidence has recentlysuggested that ART could be associated with long-term cardio-vascular changes. It was demonstrated the increased bloodpressure in late childhood after ART conception [4]. Anotherstudy demonstrated the presence of signs of systemic andpulmonary vascular dysfunction in children conceived by ART[5]. Systemic endothelial dysfunction represents the first step inthe development of atherosclerosis and is already detectable inapparently healthy children at increased cardiovascular risk [6,7].One of the consequences of the endothelial dysfunction is theincrease in vascular resistance and impedance. The isovolumicrelaxation time (IRT) has been reported to be a useful, non-invasive,Doppler-derived left ventricular (LV) relaxation index that couldserve as an index of ventricular afterload and peripheral vascularresistance [8,9]. By incorporating only time interval, the index isless dependent on anatomy or precise imaging. Furthermore, IRT isindependent of ventricular geometry [10]. We hypothesized that IRTwould be technically feasible to obtain in the embryo using thepulsed Doppler technique. By simultaneously obtaining the mitralvalve inflow waveforms and the aortic outflow waveforms from theLV outflow tract, the IRT interval could be measured.Accumulating evidence suggests that imbalance betweencirculating angiogenic factors such as vascular endothelialgrowth factor (VEGF) and anti-angiogenic factors such as solublevascular endothelial growth factor receptor-1 (sVEGFR-1) and thesoluble form of endoglin (sEng) is the central key in endothelialdysfunction and pathophysiology of preeclampsia [11].We therefore assessed stable metabolites of nitric oxide (NO),endothelin concentration, vascular pro- and anti-angiogenicgrowth factors and early embryo hemodynamics (IRT) inwomen who became pregnant as the result of ART and inwomen who conceived naturally.The hypothesis was that ART induced endothelial dysfunctionand early embryonic hemodynamic changes, which are related toepigenetic mechanisms.Materials and methodsWe conducted a prospective study utilizing a clinical pregnancy asendpoint. A total of 39 consecutive unfertile women received firstor repeated in vitro fertilization – embryo transfer treatment forAddress for correspondence: P. B. Tsyvian, Mother and Child CareResearch Institute, Russian Ministry of Public Health, Yekaterinburg,Russia. Tel: #7(343) 371-8768. E-mail: pavel.tsyvian@gmail.com123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132tubal, endometriosis and unexplained factors (ART group) in thedepartment of reproductive medicine at Mother and Child ResearchInstitute (Yekaterinburg, Russia). The control group consisted of 68women with naturally conceived pregnancy. All women had anormal body mass index (BMI, 19–23 kg/m2) and regular men-strual cycle with basal follicle stimulating hormone (FSH)510 IU/l. There was no history of ovarian operation in women of bothgroups. Patients undergoing controlled ovarian hyperstimulationwith low ovarian response (55 follicles with diameter416 mm andE251000 pg/ml on the day of hCG injection) were excluded. Also,exclusion criteria were: history of recurrent miscarriage (threeconsecutive miscarriages), distortion of the uterine cavity shown onultrasound scan and ectopic pregnancy following IVF treatment.The study was approved by institutional ethics committee and allsubjects provided written informed consent.All ART group women were pre-treated with buserelin (Suprecur,Hoechst, Frankfurt, Germany) nasal spray 150 mg four times a dayfrom the mid-luteal phase of the cycle preceding the treatment cycleand received human menopausal gonadotrophin (hMG), (Pergonal,Serono, Geneva, Switzerland) for ovarian stimulation. Humanchorionic gonadotrophin (hCG) (Profasi, Serono, Geneva,Switzerland) was given intramuscularly when the leading folliclereached 18 mm in diameter and there were at least three follicles of16 mm in diameter. Serum estradiol (E2) concentration was measuredon the day of hCG administration. Transvaginal ultrasound-guidedoocyte retrieval was scheduled 36 h after the hCG injection.All ultrasound examinations were performed using a Voluson730 Expert (GE Medical SystemsQ2 ) ultrasound system equippedwith RIC 5–9H vaginal and RAB 4–8L abdominal transducers.Ultrasonography was performed strictly adhering to the ALARA(as low as reasonably achievable) principle, and the total time ofultrasound exposure was restricted to a maximum of 20 min. Afterconfirming fetal viability and excluding the presence of anyobvious fetal anomaly, the crown-ramp length was measured.Echocardiography was performed transabdominally in all cases,and additional transvaginal examination was performed when thetransabdominal image was sub-optimal. A systematic assessment offetal heart structure was performed, obtaining standard twodimensional views [12]. Valve clicks were used to identify theclosure and opening of the atrioventricular and semilunar valveswhile measuring the time intervals [10].The LV inflow and outflowblood velocity waveforms were obtained simultaneously and theIRT (ms; time interval between the closure of the aortic valve andthe opening of the mitral valve) was measured. All the Dopplerrecordings were performed during fetal quiescence over four to sixcardiac cycles. For all the parameters assessed, an average of threeseparate measurements was used for statistical analysis).Maternal serum concentrations of VEGF, sVEGFR-1 andendothelin 1 were evaluated using commercially available ELISAkits (Bender Medsystems, AustriaQ2 ). Validation test were per-formed for serum and standard curve was obtained every time ofdetection. The concentrations of VEGF, sVEGFR-1 and endothe-lin 1 were determined by interpolation from the standard curve.All samples were examined in duplicate. The sensitivity of theELISA kits to VEGF and sVEGFR-1 was 25 and 15 pg/ml,respectively. The intra- and inter-assay coefficients of variation(CVs) for VEGF, sVEGFR-1 and endothelin 1 were both lowerthan 10%. Serum concentration of soluble Endoglin (sEng) wasassessed by R&D Systems (USA) kit.Q2 Nitrite and nitrate, thestable metabolic products of NO, were measured spectrophoto-metrically using R&D Systems (USA) kit.Statistical analysisAll data were analysed by STATISTICA 10.0 (StatSoft).Q2 Thevalues of IRT measurement data are expressed as mean ± SD.The results for pro- and anti-angiogenic factors concentrations areexpressed as median (range). Differences between the groupswere tested for significance using independent-samples t-test.Bonferroni correction was adopted for multiple comparisons.Statistical significance was defined as p50.05.ResultsTable 1 demonstrates the concentrations of the main pro- and anti-angiogenic growth factors and stable metabolites of NO. Theconcentration of pro-angiogenic agent endothelin 1 was 45.5times more in the serum of the women of ART group than incontrol. On the contrary, the concentration of the stable metab-olites of NO in ART group was 1.9 times less than in the controlgroup. There was no significant difference in the concentration ofVEGF and control groups. The assessment of angiogenesissuppressors content (endoglin and soluble receptor to VEGF –sVEGF-R1) demonstrates the decrease in concentrations of theseagents in ART group correspondently 1.6 and 2.0 times incomparison to the control group.Echocardiographic assessments of embryonic LV IRT (IRT) inART and control groups are presented in Table 2. The meanvalues of ART (ms) at 11, 12 and 13 weeks of gestation weresignificantly less in ART group in comparison with the controlgroup.DiscussionThe significant increase in one of the strongest vasoconstrictors –endothelin 1 and concomitant decrease in vasodilation agentssynthesis (NO and its metabolites) in ART group was demon-strated in this study. The decrease in synthesis of angiosuppres-sors (endoglin and sVEGF-R1) could reflect the down-regulationof these agents as a result of general vasoconstrictive reaction inART women during early pregnancy. We also demonstrated asignificant increase in embryonic LV IRT in ART group. Thisfinding can reflect the increase in vascular impedance at this earlystage of ART embryos development.Due to young age of the ART population in humans, it is notknown yet whether ART is associated with increased risk forclinical cardiovascular endpoints. However, there is abundantevidence that in population at risk, atherosclerosis and cardio-vascular diseases already start in childhood many years before thefirst clinical events occur [6,7]. We propose the endothelialdysfunction as a main mechanism of changes that we observed inmaternal serum angiogenic – anti-angiogenic agents balance andIRT changes in embryos of ART group.There are several facts obtained from studies in ART micewhich could support this idea. It was shown that in ART mice,endothelium-dependent mesenteric artery dilation was defectiveand carotid artery stiffness was increased [13]. In ART, thisdefective vascular function in vitro was translated into significantarterial hypertension in vivo [13].In humans, it is difficult to completely exclude that parentalfactors contribute to vascular dysfunction in ART children. Thefindings that in normal mice ART induces premature vascularaging and arterial hypertension, however, provide strong add-itional evidence for the concept that ART per se is the main causeof the observed changes. The findings in mice also strengthen theconcept that hormonal stimulation of the ovulation in the motheris not important determinant of ART-induced vascular dysfunc-tion, because endothelium-dependent vasodilation of mesentericartery was normal in offspring of super-ovulated mice [14].In offspring of mice with protein-restricted diet duringpregnancy, pulmonary vascular dysfunction is associated withaltered lung DNA methylation suggesting that epigenetic mech-anism may be involved in the fetal programming of the vascular2 N. V. Bashmakova et al. Gynecol Endocrinol, Early Online: 1–3133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211212213214215216217218219220221222223224225226227228229230231232233234235236237238239240241242243244245246247248249250251252253254255256257258259260261262263264system [15]. Moreover, it was demonstrated that epigeneticalteration may participate in ART mice vascular changes. It wasfound that the methylation of the promoter of the gene coding forendothelial nitric oxide synthase (eNOS) was altered in the aortaof ART mice [15]. This demethylation had important conse-quences, as evidence by decreased eNOS and eNOS RNAexpression in the vascular bed and impaired vascular NOsynthesis in ART compared with control mice [15].Declaration of interestThis study was supported by a grant from the Russian Foundation forBasic Research-Ural (No: 13-04-96080). The authors report no declar-ations of interest.References1. Sutcliffe AG, Ludwig M. Outcome of assisted reproduction. Lancet2007;370:351–19.2. Barker DJ. The origins of the developmental origins theory. J InternMed 2007;261:412–17.3. Celermajer DS. Manipulating nature: might there be a cardiovas-cular price to pay for the miracle of assisted conception? Circulation2012;125:1832–4.4. Ceelen M, van Weissenbruch MM, Vermeiden JP, et al.Cardiometabolic differences in children born after in vitro fertiliza-tion: follow-up study. J Clin Endocrinol Metab 2008;93:1682–8.5. Scherrer U, Rimoldi SF, Rexhaj E, et al. Systemic and pulmonaryvascular dysfunction in children conceived by assisted reproductivetechnologies. Circulation 2012;125:1890–6.6. Charakida M, Deanfielf JE, Halcox JP. Childhood origins of arterialdisease. Curr Opin Pediatr 2007;19:538–45.7. Urbina EM, Williams RV, Alpert BS, et al. Noninvasive assessmentof subclinical atherosclerosis in children and adolescents: recom-mendations for standard assessment fo9r clinical research: ascientific statement from American Heart Association.Hypertension 2007;54:919–50.8. Tsyvian P, Malkin K, Wladimiroff JW. Assessment of mitral A-wavetransit time to cardiac outflow tract and isovolumic relaxation timeof left ventricle in the appropriate and small for gestational agehuman fetus. Ultrasound Med Biol 1997;23:187–90.9. Tsyvian P, Markova TV, Mikhailova SV, et al. Left ventricularisovolumic relaxation and rennin-angiotensin system in the growthrestricted fetus. Europ J Obstet Gyn Reprod Biol 2008;140:33–7.10. Eidem BW, O’Leary P, Tei C, Seward J. Usefulness of themyocardial performance index for assessing right ventricularfunction in congenital heart disease. Am J Cardiol 2000;86:654–8.11. Levine RJ, Maynard SE, Qian C, et al. Circulating angiogenicfactors and the risk of preeclampsia. N Engl J Med 2004;350:672–83.12. Gembruch U, Shi C, Smrcek J. Biometry of the fetal heart between10 and 17 weeks of gestation. Fet Diagn Ther 2000;15:20–31.13. Rexaj E, Paoloni-Giacobino A, Rimoldi SF, Fuster DG. Micegenerated by in vitro fertilization exhibit vascular dysfunction andshortened life span. J Children Invest 2013;123:5052–6010.14. Rexaj E, Bloch J, Jayet PY, et al. Fetal programming of pulmonaryvascular dysfunction in mice: role of epigenetic mechanisms. Am JPhysiol Heart Circ Physiol 2011;301:H247–52.15. Rimoldi SF, Sartori C, Rexhaj E, et al. Vascular dysfunction inchildren conceived by assisted reproductive technologies: under-lying mechanism and future implications. Swiss Med Wkly 2014;144:1–11.Table 1. Serum concentrations of pro- and anti-angiogenic agents in maternal groups.Analyte ART group (n¼ 39) Control group (n¼ 68) p ValueEndothelin 1 (fmol/l) 1.82 (0.63–3.11) 0.04 (0.004–0.45) 0.0001VEGF, pg/ml 0.14 (0.0–5.16) 0.09 (0.0–0.66) n.s.sVEGFR-1, pg/ml 0.54 (0.03–1.08) 1.06 (0.46–1.96) 0.01NO2 gen., mkM/l 16.38 (14.23–21.83) 19.35 (15.24–26.26)Q3NO2 end., mkM/l 1.66 (1.09–2.34) 3.18 (1.26–5.16) 0.004NO3, mkM/l 14.56 (12.42–19.65) 16.07 (11.92–20.7) n.s.sEndoglin, ng/ml 5.31 (4.61–6.19) 8.56 (6.99–10.41) 0.0001The results are expressed as median (range).Table 2. Left ventricular isovolumic relaxation time (ms) values inmaternal groups.Gestational age (wk) ART group Control group p Value11 35 ± 3 27 ± 2 0.0112 37 ± 3 28 ± 3 0.0113 39 ± 3 30 ± 2 0.01DOI: 10.3109/09513590.2015.1085199 Endothelial function and vascular impedance in ART embryos 3265266267268269270271272273274275276277278279280281282283284285286287288289290291292293294295296297298299300301302303304305306307308309310311312313314315316317318319320321322323324325326327328329330331332333334335336337338339340341342343344345346347348349350351352353354355356357358359360361362363364365366367368369370371372373374375376377378379380381382383384385386387388389390391392393394395396

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Endothelial function, regulation of angiogenesis and embryonic central hemodynamics in ART-conceived pregnancies

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Endothelial function, regulation of angiogenesis and embryonic central hemodynamics in ART-conceived pregnancies

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