- Email: [email protected]
Intrauterine growth restriction and circulating neurotrophin levels at term
Early Human Development (2007) 83, 465–469
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / e a r l h u m d e v
Intrauterine growth restriction and circulating neurotrophin levels at term Ariadne Malamitsi-Puchner ⁎, Konstantinos E. Nikolaou, Emmanuel Economou, Maria Boutsikou, Theodora Boutsikou, Marialena Kyriakakou, Karl-Philipp Puchner, Demetrios Hassiakos Neonatal Division, Second Department of Obstetrics and Gynecology, University of Athens, Athens, Greece Accepted 1 September 2006
KEYWORDS Neurotrophins; Intrauterine growth restriction; Perinatal period; Fullterm neonates; Brain sparing effect
Abstract Background: Intrauterine growth restricted (IUGR) fetuses are those with estimated weight b 10th customized centile, displaying signs of chronic malnutrition and hypoxia leading to brain sparing effect. Neurotrophins, [Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4 (NT-4)] are important for pre- and post-natal brain development. Aims: To investigate circulating NGF, BDNF, NT-3 and NT-4 levels in IUGR and appropriate for gestational age (AGA) fullterm fetuses and neonates (day-1 [N1] and day-4 [N4]) and in their mothers. Study design: Prospective case control study. Subjects: 60 mothers and their single 30 IUGR and 30 AGA fullterm fetuses and neonates. Outcome measures: Determination, by enzyme immunoassays, of NGF, BDNF, NT-3 and NT-4 plasma levels. Results: No statistically significant differences existed between IUGR and AGA maternal, fetal and neonatal levels of BDNF, NT-3 and NT-4. NGF was significantly higher in AGA than IUGR maternal (p = 0.007), fetal (p = 0.01), neonatal day 1 (p = 0.043) and 4 (p = 0.003) plasma, and positively correlated with the infants' centiles and birthweights. IUGR and AGA maternal neurotrophins were higher than the respective fetal and neonatal ones and no correlation with gender or delivery mode in both groups was observed. Conclusions: In the perinatal period, circulating levels of BDNF, NT-3 and NT-4 do not differ in IUGR and AGA pregnancies, in contrast to NGF levels, which are higher in the AGA group. NGF is the only neurotrophin correlating with customized centiles and birthweights of the infants. Neurotrophin concentrations are higher in maternal plasma and do not depend on gender. © 2006 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: AGA, appropriate for gestational age; BDNF, brain derived neurotrophic factor; IUGR, intrauterine growth restriction; MS, maternal sample; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; N1, neonatal day 1 sample; N4, neonatal day 4 sample; UC, umbilical cord sample. ⁎ Corresponding author. Neonatal Division, Second Department of Obstetrics and Gynecology, University of Athens, 19, Soultani str., GR10682, Athens, Greece. Tel.: +30 6944 443815; fax: +30 210 7233330. E-mail addresses: [email protected], [email protected] (A. Malamitsi-Puchner). 0378-3782/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2006.09.001
466
1. Introduction The neurotrophin family comprises four structurally related molecules: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) [1]. These factors share a greater than 80% identity at the amino acid level and mediate their effects via two types of receptors: the high affinity tyrosine kinase (Trk) receptors A (NGF), Trk B (BDNF and NT-4) and Trk C (NT-3) [2] and the low affinity p75 receptor, which is a member of the tumor necrosis factor receptor family[3]. Neurotrophins are neuroprotective factors, reducing apoptosis and promoting survival and maintenance of specific populations of neurons in both the peripheral (sympathetic and sensory neurons) and the central (cholinergic, dopaminergic, adrenergic neurons) nervous systems [2,4–6]. They are important for axon growth during development [7], higher neuronal functions [8], developmental maturity of the cortex and synaptic plasticity, leading to refinement of connections [9], morphologic differentiation and neurotransmitter expression [10]. Thus, neurotrophins play an important role in prenatal and post-natal brain development [11]. Intrauterine growth restriction (IUGR) characterizes the fetus that fails to reach its intrinsic growth potential and is at increased risk of perinatal morbidity and mortality [1,12]. Factors determining the intrinsic growth potential include: maternal height and booking weight, ethnicity, parity, gender, number of in utero coexisting fetuses and altitude at which pregnancy evolves [13–15]. Asymmetrical IUGR fetuses are those with an estimated weight below the 10th customized centile [14], who are not constitutionally small, and display signs of chronic malnutrition and hypoxia [16], mainly due to deficient placental transport of nutrients and oxygen [17]. Chronic hypoxia urges the fetus to redistribute blood flow to the most important organs, such as the brain, myocardium and adrenal glands, while other organs (e.g. the kidneys) are hypoperfused. This phenomenon has been called the brain sparing effect and is regularly accompanied by oligohydramnios [18,19]. Considering the brain sparing effect, this study was based on the hypothesis that circulating neurotrophin levels (eventually reflecting central levels and, thus, cerebral status) should not differ between growth restricted and normal fullterm infants. Therefore, we aimed to determine circulating levels of NGF, BDNF, NT-3 and NT-4 in IUGR and appropriate for gestational age (AGA) fullterm infants at time-points characteristic for intra- and extrauterine life.
2. Material and methods The study was approved by the Ethics Committee of our teaching hospital and informed consent was obtained from participating mothers. We included in this study – which lasted from April 2004 to April 2005 – thirty asymmetrical IUGR and 30 AGA fullterm neonates, consecutively enrolled, as well as their mothers. As IUGR were characterized infants below the 10th customized centile for birth weight. For the calculation of the customized centiles, the principles of the Gestation Related Optimal Weight (GROW) program [13,14]
A. Malamitsi-Puchner et al.
Table 1 Demographic data of participating intrauterine growth restricted (IUGR) (n = 30) and appropriate for gestational age (AGA) (n = 30) fetuses and newborns), as well as of their mothers (n = 60) IUGR AGA p (mean ± SD) (mean ± SD) value Centile Gestational age (weeks) Placental weight (g) Maternal age (years) Maternal height (cm) Maternal booking weight (kg) Birth-weight (g) Birth length (cm) Head circumference (cm) Umbilical artery pH Male/female n (%) Cesarean section n (%) Umbilical cord artery Pulsatility index (PI) Umbilical cord artery Resistance index (RI)
4.7 ± 3.3 38.3 ± 1.4
37.2 ± 18.8 38.4 ± 1.1
b0.001 0.739
450.8 ± 100.2 28.3 ± 5.2
603.7 ± 153.1 31.1 ± 4.4
b0.001
163.5 ± 7.0
166.0 ± 6.2
0.159
61.4 ± 12.5
60.6 ± 8.7
0.806
2484 ± 334 48.2 ± 2.2 33.2 ± 1.3
3081 ± 328 50.4 ± 2.1 34.8 ± 1.7
b0.001 b0.001 b0.001
7.26 ± 0.02 22 (73.7)/ 8 (26.7) 17 (57.1%)
7.36 ± 0.07 16 (53.3)/ 14 (46.7) 19 (63.3%)
b0.001 0.09
0.76 ± 0.14
0.79 ± 0.12
0.27
0.54 ± 0.85
0.53 ± 0.84
0.72
0.028
0.416
were used. This is a computer-generated antenatal chart that can be customized for each pregnancy, taking into consideration significant determinants of birthweight, such as gestational age, neonatal gender, maternal weight at the beginning of pregnancy, maternal height, ethnic group and parity. These parameters are entered into the program to adjust the normal birthweight centile limits [13]. The cause of intrauterine growth restriction was identified in each of our 30 IUGR neonates. The personal, family and perinatal histories of each parturient were evaluated. Every 10–15 days, starting from the 32nd week of gestation, fetal ultrasounds and Doppler studies of the maternal uterine arteries, as well as of the umbilical and middle cerebral arteries of the fetuses, were performed. Pulsatility index (PI) was defined as the difference between peak systolic (S) and end diastolic (D) frequencies divided by the mean Doppler shift, and resistance index (RI) was defined as the peak systolic frequency minus the end diastolic frequency divided by the peak systolic frequency. For the evaluation of the amniotic fluid, the largest fluid column on the vertical plane was assessed. When it was b 2 cm, the volume was defined as diminished. All mothers were checked for congenital infections and were found negative; those over 35 years had undergone prenatal chromosomal studies, which were normal. Asymmetrical IUGR was associated with preeclampsia in 5 cases, with gestational hypertension in 10 cases, and with chronic diseases (severe anaemia, type I diabetes mellitus,
Intrauterine growth restriction and circulating neurotrophin levels at term
467
4. Results
Figure 1 Median values of NGF in maternal (MS), fetal (UC) and neonatal plasma on day 1 (N1) and 4 (N4) postpartum of intrauterine growth restricted (IUGR) and appropriate for gestational age (AGA) groups.
hepatitis B, rheumatoid arthritis, renal insufficiency, asthma and psoriasis) in the remaining cases; all of the above resulted in small and infarcted placentas. Five mothers reported smoking 10 cigarettes per day. In the group of AGA infants, mothers were healthy; one smoked up to 5 cigarettes per day and another reported daily consumption of one glass of beer. All placentas in the AGA group were normal in appearance and weight. The neonates included in the study were single and did not present congenital infections or genetic syndromes. One- and fiveminute Apgar scores were in all IUGR cases and AGA controls N 7 and N 8, respectively. Demographic data of the participating infants, as well as of their mothers, are given in Table 1. Blood was drawn from the mothers at the first stage of labor or before receiving anesthesia in cases of elective cesarean section, from the doubly clamped umbilical cord. Blood was drawn from the neonates on days 1 (N1) and 4 (N4), characteristic of transition and stabilization to extrauterine life, respectively. Collected blood (240 samples in total) was centrifuged and supernatant plasma was kept frozen at − 80 °C until assay. The determination of all neurotrophins was performed by enzyme immunoassays [human β-NGF, Catalog Number DY256; human BDNF, Catalog Number DY248; human NT-3, Catalog Number DY267; human NT-4, Catalog Number DY268, (R&D Systems Inc, Minneapolis, MN 55413, USA)]. Minimum detectable concentration, intra- and inter-assay coefficients of variation were: for β-NGF 31.3 pg/mL, 8.9% and 12.7%, respectively; for BDNF 20 pg/mL, 7.5% and 11.3%, respectively; for NT-3 40.3 pg/mL, 8.2% and 11.9%, respectively; and for NT-4 25.8 pg/mL, 10.5%, and 15%, respectively.
3. Statistical analysis All neurotrophin data, except BDNF data, were not normally distributed (Kolmogorov–Smirnov test). Therefore, nonparametric procedures (Mann Whitney U test, Wilcoxon sum rank test, Spearman Rank correlation test) were applied in the statistical analysis as appropriate. P values b 0.05 were considered significant.
Amniotic fluid was diminished in all IUGR cases. IUGR and AGA fetuses did not present any differences in the umbilical cord artery pulsatility index (PI) and resistance index (RI), as demonstrated in Table 1. No statistically significant differences were observed among IUGR and AGA groups in maternal, fetal and neonatal day-1 and day-4 samples concerning levels of neurotrophins BDNF, NT-3 and NT-4. However, NGF levels were significantly higher in AGA maternal (p = 0.007), fetal (p = 0.01), and neonatal day-1 (p = 0.043) and day-4 (p = 0.003) samples as compared to respective levels in IUGR samples (Fig. 1). NGF levels in maternal, fetal and neonatal day-1 and day-4 samples positively correlated to the infants' centiles (p ranging from 0.04 to 0.0004) and birthweights (p ranging from 0.015 to 0.003). Nevertheless, BDNF, NT-3 and NT-4 levels neither in maternal, fetal or neonatal samples showed similar correlations with the infants' centiles and birthweights. Maternal levels were, in both IUGR and AGA groups, higher than respective fetal and neonatal levels (for all cases, p ranged from p b 0.001 to p = 0.04). Lastly, determined neurotrophin levels showed no correlation with gender or mode of delivery in both IUGR and AGA groups.
5. Discussion The results of this study indicate that no statistically significant differences were observed between fullterm IUGR and AGA groups concerning respective maternal, fetal and neonatal plasma levels of BDNF, NT-3 and NT-4. In contrast, NGF levels were significantly higher in all samples of the AGA group as compared to the IUGR group. During the perinatal period the blood–brain barrier is immature and, thus, circulating levels of neurotrophins may represent respective CNS levels [20]. Relatively, circulating BDNF levels have been previously shown to correlate with cortical BDNF levels [21]. Asymmetric IUGR cases are characterized by in utero hypoxia, which has been shown to influence a series of factors, mainly involved in angiogenesis, including angiopoietin-2 [22–24], vascular endothelial growth factor and placenta growth factor [25,26], endostatin [27,28] and soluble vascular endothelial growth factor receptor-1 [25,29]. Nevertheless, according to this study, in asymmetric fullterm IUGR infants probable intrauterine hypoxia does not seem to influence circulating – and presumably central – neurotrophin levels, a fact possibly implying the lack of CNS compromise, as neurotrophins are implicated in nervous system physiology. It is known that growth restriction successfully balances reduced oxygen delivery and consumption [30]. Thus, by restricting growth, the fetal organism manages to maintain or to minimally decrease arterial oxygen concentration, until substrate delivery is severely reduced [31]. However, minor reduction in overall oxygen consumption may mask large organ differences that, to some extent, mirror the pattern of blood flow redistribution [30]. As a consequence, cerebral metabolism is preserved, while oxygen consumption in
468 several organs, including the kidneys, is markedly decreased [30]. In reference to the latter, one can explain the diminished amniotic fluid [32], also observed in the present study. Although the mean customized centile of the asymmetric IUGR infants included in this study was considerably below the 10th customized centile, they did not present detectable changes in the umbilical and middle cerebral artery fetal Doppler studies and had an uncomplicated transition and stabilization to extrauterine life. In respect to this, it should be stressed that all infants included in the study reached term, thus growth restriction developed late in the third trimester, and therefore had a less adverse effect on the outcome than if it were initiated at an earlier stage of pregnancy, at a time when the brain might be more vulnerable [33] and preterm delivery would be unavoidable [34]. However, as already stated, all IUGR pregnancies were characterized by oligohydramnios, reflecting kidney hypoperfusion and blood redistribution (brain sparing effect) [18,12]. As for the deviating results of NGF, it has been shown that NGF levels measured in the umbilical cord of preterm infants were significantly lower than levels determined in fullterms [35]. In addition, we have also previously reported [36] that BDNF fetal and neonatal plasma concentrations were significantly higher in fullterm as compared to preterm cases. Although all IUGR infants included in this study were fullterms, one should take into consideration that they had a significantly lower birthweight than AGA infants, as prematures also have. Relatively, the present study showed that fetal and neonatal NGF plasma levels positively correlated with the centile and the birthweight of the infant, thus justifying higher NGF levels in the AGA group. It is noteworthy that this positive correlation did not apply to any other neurotrophin. On the other hand, Korhonen et al. [37] demonstrated that in infants with asphyxia, cerebrospinal fluid levels of NGF were significantly decreased as compared to the respective levels of infants without asphyxia. It could be speculated that similar findings are expected for NGF plasma levels, also in cases of in utero hypoxia. Although the umbilical artery pH values were statistically lower in the IUGR group compared to the AGA group in our study, they were not clinically significant, as all umbilical artery pH values were within normal limits. Furthermore, our study showed that in both IUGR and AGA groups, fetal and neonatal neurotrophin levels are lower than the respective maternal levels. This fact, confirming previous reports (referring only to BDNF in normal pregnancies, [36]) could be interpreted by taking into account the more mature peripheral and central nervous system of adults, given the developmental appearance of neurotrophins in humans [35]. Finally, this study did not find any statistical difference in neurotrophin levels both in IUGR and AGA groups between girls and boys [19,36]. Therefore, we can assume that neurodevelopment was similar in both genders. In conclusion, in the perinatal period, circulating levels of neurotrophins BDNF, NT-3 and NT-4 do not differ in IUGR and AGA pregnancies. In contrast, circulating NGF levels are higher in the AGA group; furthermore, NGF is the only neurotrophin for which the circulating levels are found to correlate with the customized centiles and the birthweights of the infants. Maternal neurotrophin levels are higher than
A. Malamitsi-Puchner et al. fetal or neonatal ones. Lastly, neither gender nor mode of delivery influence circulating neurotrophin levels in both IUGR and AGA groups at term. Further studies should investigate circulating neurotrophin levels in IUGR preterm infants, as well as in those with pronounced cerebral insults, manifested in prenatal blood flow Doppler studies.
References [1] Barde Y-A. The nerve growth factor family. Prog Growth Factor Res 1990;2:237–48. [2] Lewin G, Barde Y-A. Physiology of the neurotrophins. Annu Rev Neurosci 1996;19:289–317. [3] Barbacid M. Neurotrophic factors and their receptors. Curr Opin Cell Biol 1995;7:148–55. [4] Arenas E, Persson H. Neurotrophin-3 prevents the death of adult central noradrenergic neurons in vivo. Nature 1994;367: 368–71. [5] Hefti F. Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections. J Neurosci 1986;6:2155–62. [6] Hyman C, Hofer M, Barde YA, Juhasz M, Yancopoulos GD, Squinto S, et al. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 1991;350:230–2. [7] Tucker KL, Meyer M, Barde YA. Neurotrophins are required for nerve growth during development. Nat Neurosci 2001;4:29–37. [8] Chao MV. Trophic factors: an evolutionary cul-de-sac or door into higher neuronal function? J Neurosci Res 2001;59:353–5. [9] Lu B, Figurov A. Role of neurotrophins in synapse development and plasticity. Rev Neurosci 1997;8:1-12. [10] Takei N, Nawa H. Roles of neurotrophins on synaptic development and functions in the central nervous system. Hum Cell 1998;11:157–65. [11] Chouthai NS, Sampers J, Desai N, Smith GM. Changes in neurotrophin levels in umbilical cord blood from infants with different gestational ages and clinical conditions. Pediatr Res 2003;53:965–9. [12] Resnik R. Intrauterine growth restriction. Obstet Gynecol 2002;99:490–6. [13] Gardosi J, Chang A, Kalyan B, Sahota D, Symonds EM. Customised antenatal growth charts. Lancet 1992;339:283–7. [14] Gardosi J, Mongelli M, Wilcox M, Chang A. An adjustable fetal weight standard. Ultrasound Obstet Gynecol 1995;6:168–74. [15] Moore LG, Shriver M, Bemis L, Hickler B, Wilson M, Brutsaert T, et al. Maternal adaptation to high-altitude pregnancy: an experiment of nature—a review. Placenta 2004;25(Suppl A): S60–71. [16] Ville Y, Nyberg DA. Growth, Doppler and fetal assessment. In: Nyberg DA, McGahan JP, Pretorius DH, Pilu G, editors. Diagnostic imaging of fetal anomalies. Philadelphia: Lippincott, Williams and Wilkins; 2003. p. 31–58. [17] Soothill PW, Ajayi RA, Nicolaides KN. Fetal biochemistry in growth retardation. Early Hum Dev 1992;29:91–7. [18] Dubiel M, Seremak-Mrozikiewicz A, Breborowicz GH, Drews K, Pietryga M, Gudmundsson S. Fetal and maternal Doppler velocimetry and cytokines in high-risk pregnancy. J Perinat Med 2005;33(1):17–21. [19] Harkness UF, Mari G. Diagnosis and management of intrauterine growth restriction. Clin Perinatol 2004;31:743–64. [20] Polin RA, Fox WW. Fetal and neonatal physiology. Philadelphia: WB Saunders; 1998. [21] Karege F, Schwald M, Cisse M. Postnatal developmental profile of brain derived neurotrophic factor in rat brain and platelets. Neurosci Lett 2002;328:261–4. [22] Krikun G, Schatz F, Finlay T, Kadner S, Mesia A, Gerrets R, et al. Expression of angiopoietin-2 by human endometrial endothelial cells: regulation by hypoxia and inflammation. Biochem Biophys Res Commun 2000;275:159–63.
Intrauterine growth restriction and circulating neurotrophin levels at term [23] Malamitsi-Pucchner A, Boutsikou T, Economou E, Tzonou A, Makrakis E, Nikolaou KE, Hassiakos D. Angiopoietin-2 in the perinatal period and the role of intrauterine growth restriction. Acta Obstetricia et Gynecogica Scand 2006;85:45–8. [24] Yuan HT, Yang SP, Woolf AS. Hypoxia up-regulates angiopoietin-2, a Tie-2 ligand, in mouse mesangial cells. Kidney Int 2000;58:1912–9. [25] Ahmed A, Dunk C, Ahmad S, Khaliq A. Regulation of placental vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) and soluble Flt-1 by oxygen—A review. Placenta; 21: Supp. A, Trophoblast Research 14: (2000) S16–S24. [26] Malamitsi-Puchner A, Boutsikou T, Economou E, Sarandakou A, Makrakis E, Hassiakos D, et al. Vascular endothelial growth factor and placenta growth factor in intrauterine growthrestricted fetuses and neonates. Mediat Inflamm 2005;5:293–7. [27] Malamitsi-Puchner A, Boutsikou T, Economou E, Makrakis E, Iliodromiti Z, Kouskouni E, et al. The role of the anti-angiogenic factor endostatin in intrauterine growth restriction. J Soc Gynecol Investig 2005;12:195–7. [28] Wu P, Yonekura H, Li H, Nozaki I, Tomono Y, Naito I, et al. Hypoxia down-regulates endostatin production by human microvascular endothelial cells and pericytes. Biochem Biophys Res Commun 2001;288:1149–54. [29] Boutsikou T, Malamitsi-Puchner A, Economou E, Boutsikou M, Puchner K-P, Hassiakos D. Soluble vascular endothelial growth
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factor receptor-1 in intrauterine growth restricted fetuses and neonates. Early Hum Dev 2006;82:235–9. Peebles DM. Fetal consequences of chronic substrate deprivation. Semin Fetal Neonatal Med 2004;9:379–86. Richardson BS, Bocking AD. Metabolic and circulatory adaptations to chronic hypoxia in the fetus. Comp Biochem Physiol 1998;119A:717–23. Braems G, Jensen A. Hypoxia reduces oxygen consumption of fetal skeletal muscle cells in monolayer culture. J Dev Physiol 1991;16:209–15. Dobbing J. The later development of the brain and its vulnerability. In: Davis JA, Dobbing J, editors. Scientific foundations of paediatrics. 2nd ed. London: Heinemann; 1981. Roth S, Chang TC, Robson S, Spencer JA, Wyatt JS, Stewart AL. The neurodevelopmental outcome of term infants with different intrauterine growth characteristics. Early Hum Dev 1999;55:39–50. Haddad J, Vilge V, Juif JG, Maitre M, Donato L, Messer J, et al. Beta-nerve growth factor levels in newborn cord sera. Pediatr Res 1994;35:637–9. Malamitsi-Puchner A, Economou E, Rigopoulou O, Boutsikou T. Perinatal changes of brain-derived neurotrophic factor in preand fullterm neonates. Early Hum Dev 2004;76:17–22. Korhonen L, Riikonen R, Nawa H, Lindholm D. Brain derived neurotrophic factor is increased in cerebrospinal fluid of children suffering from asphyxia. Neurosci Lett 1998;16:151–4.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / e a r l h u m d e v
Intrauterine growth restriction and circulating neurotrophin levels at term Ariadne Malamitsi-Puchner ⁎, Konstantinos E. Nikolaou, Emmanuel Economou, Maria Boutsikou, Theodora Boutsikou, Marialena Kyriakakou, Karl-Philipp Puchner, Demetrios Hassiakos Neonatal Division, Second Department of Obstetrics and Gynecology, University of Athens, Athens, Greece Accepted 1 September 2006
KEYWORDS Neurotrophins; Intrauterine growth restriction; Perinatal period; Fullterm neonates; Brain sparing effect
Abstract Background: Intrauterine growth restricted (IUGR) fetuses are those with estimated weight b 10th customized centile, displaying signs of chronic malnutrition and hypoxia leading to brain sparing effect. Neurotrophins, [Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4 (NT-4)] are important for pre- and post-natal brain development. Aims: To investigate circulating NGF, BDNF, NT-3 and NT-4 levels in IUGR and appropriate for gestational age (AGA) fullterm fetuses and neonates (day-1 [N1] and day-4 [N4]) and in their mothers. Study design: Prospective case control study. Subjects: 60 mothers and their single 30 IUGR and 30 AGA fullterm fetuses and neonates. Outcome measures: Determination, by enzyme immunoassays, of NGF, BDNF, NT-3 and NT-4 plasma levels. Results: No statistically significant differences existed between IUGR and AGA maternal, fetal and neonatal levels of BDNF, NT-3 and NT-4. NGF was significantly higher in AGA than IUGR maternal (p = 0.007), fetal (p = 0.01), neonatal day 1 (p = 0.043) and 4 (p = 0.003) plasma, and positively correlated with the infants' centiles and birthweights. IUGR and AGA maternal neurotrophins were higher than the respective fetal and neonatal ones and no correlation with gender or delivery mode in both groups was observed. Conclusions: In the perinatal period, circulating levels of BDNF, NT-3 and NT-4 do not differ in IUGR and AGA pregnancies, in contrast to NGF levels, which are higher in the AGA group. NGF is the only neurotrophin correlating with customized centiles and birthweights of the infants. Neurotrophin concentrations are higher in maternal plasma and do not depend on gender. © 2006 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: AGA, appropriate for gestational age; BDNF, brain derived neurotrophic factor; IUGR, intrauterine growth restriction; MS, maternal sample; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; N1, neonatal day 1 sample; N4, neonatal day 4 sample; UC, umbilical cord sample. ⁎ Corresponding author. Neonatal Division, Second Department of Obstetrics and Gynecology, University of Athens, 19, Soultani str., GR10682, Athens, Greece. Tel.: +30 6944 443815; fax: +30 210 7233330. E-mail addresses: [email protected], [email protected] (A. Malamitsi-Puchner). 0378-3782/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2006.09.001
466
1. Introduction The neurotrophin family comprises four structurally related molecules: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) [1]. These factors share a greater than 80% identity at the amino acid level and mediate their effects via two types of receptors: the high affinity tyrosine kinase (Trk) receptors A (NGF), Trk B (BDNF and NT-4) and Trk C (NT-3) [2] and the low affinity p75 receptor, which is a member of the tumor necrosis factor receptor family[3]. Neurotrophins are neuroprotective factors, reducing apoptosis and promoting survival and maintenance of specific populations of neurons in both the peripheral (sympathetic and sensory neurons) and the central (cholinergic, dopaminergic, adrenergic neurons) nervous systems [2,4–6]. They are important for axon growth during development [7], higher neuronal functions [8], developmental maturity of the cortex and synaptic plasticity, leading to refinement of connections [9], morphologic differentiation and neurotransmitter expression [10]. Thus, neurotrophins play an important role in prenatal and post-natal brain development [11]. Intrauterine growth restriction (IUGR) characterizes the fetus that fails to reach its intrinsic growth potential and is at increased risk of perinatal morbidity and mortality [1,12]. Factors determining the intrinsic growth potential include: maternal height and booking weight, ethnicity, parity, gender, number of in utero coexisting fetuses and altitude at which pregnancy evolves [13–15]. Asymmetrical IUGR fetuses are those with an estimated weight below the 10th customized centile [14], who are not constitutionally small, and display signs of chronic malnutrition and hypoxia [16], mainly due to deficient placental transport of nutrients and oxygen [17]. Chronic hypoxia urges the fetus to redistribute blood flow to the most important organs, such as the brain, myocardium and adrenal glands, while other organs (e.g. the kidneys) are hypoperfused. This phenomenon has been called the brain sparing effect and is regularly accompanied by oligohydramnios [18,19]. Considering the brain sparing effect, this study was based on the hypothesis that circulating neurotrophin levels (eventually reflecting central levels and, thus, cerebral status) should not differ between growth restricted and normal fullterm infants. Therefore, we aimed to determine circulating levels of NGF, BDNF, NT-3 and NT-4 in IUGR and appropriate for gestational age (AGA) fullterm infants at time-points characteristic for intra- and extrauterine life.
2. Material and methods The study was approved by the Ethics Committee of our teaching hospital and informed consent was obtained from participating mothers. We included in this study – which lasted from April 2004 to April 2005 – thirty asymmetrical IUGR and 30 AGA fullterm neonates, consecutively enrolled, as well as their mothers. As IUGR were characterized infants below the 10th customized centile for birth weight. For the calculation of the customized centiles, the principles of the Gestation Related Optimal Weight (GROW) program [13,14]
A. Malamitsi-Puchner et al.
Table 1 Demographic data of participating intrauterine growth restricted (IUGR) (n = 30) and appropriate for gestational age (AGA) (n = 30) fetuses and newborns), as well as of their mothers (n = 60) IUGR AGA p (mean ± SD) (mean ± SD) value Centile Gestational age (weeks) Placental weight (g) Maternal age (years) Maternal height (cm) Maternal booking weight (kg) Birth-weight (g) Birth length (cm) Head circumference (cm) Umbilical artery pH Male/female n (%) Cesarean section n (%) Umbilical cord artery Pulsatility index (PI) Umbilical cord artery Resistance index (RI)
4.7 ± 3.3 38.3 ± 1.4
37.2 ± 18.8 38.4 ± 1.1
b0.001 0.739
450.8 ± 100.2 28.3 ± 5.2
603.7 ± 153.1 31.1 ± 4.4
b0.001
163.5 ± 7.0
166.0 ± 6.2
0.159
61.4 ± 12.5
60.6 ± 8.7
0.806
2484 ± 334 48.2 ± 2.2 33.2 ± 1.3
3081 ± 328 50.4 ± 2.1 34.8 ± 1.7
b0.001 b0.001 b0.001
7.26 ± 0.02 22 (73.7)/ 8 (26.7) 17 (57.1%)
7.36 ± 0.07 16 (53.3)/ 14 (46.7) 19 (63.3%)
b0.001 0.09
0.76 ± 0.14
0.79 ± 0.12
0.27
0.54 ± 0.85
0.53 ± 0.84
0.72
0.028
0.416
were used. This is a computer-generated antenatal chart that can be customized for each pregnancy, taking into consideration significant determinants of birthweight, such as gestational age, neonatal gender, maternal weight at the beginning of pregnancy, maternal height, ethnic group and parity. These parameters are entered into the program to adjust the normal birthweight centile limits [13]. The cause of intrauterine growth restriction was identified in each of our 30 IUGR neonates. The personal, family and perinatal histories of each parturient were evaluated. Every 10–15 days, starting from the 32nd week of gestation, fetal ultrasounds and Doppler studies of the maternal uterine arteries, as well as of the umbilical and middle cerebral arteries of the fetuses, were performed. Pulsatility index (PI) was defined as the difference between peak systolic (S) and end diastolic (D) frequencies divided by the mean Doppler shift, and resistance index (RI) was defined as the peak systolic frequency minus the end diastolic frequency divided by the peak systolic frequency. For the evaluation of the amniotic fluid, the largest fluid column on the vertical plane was assessed. When it was b 2 cm, the volume was defined as diminished. All mothers were checked for congenital infections and were found negative; those over 35 years had undergone prenatal chromosomal studies, which were normal. Asymmetrical IUGR was associated with preeclampsia in 5 cases, with gestational hypertension in 10 cases, and with chronic diseases (severe anaemia, type I diabetes mellitus,
Intrauterine growth restriction and circulating neurotrophin levels at term
467
4. Results
Figure 1 Median values of NGF in maternal (MS), fetal (UC) and neonatal plasma on day 1 (N1) and 4 (N4) postpartum of intrauterine growth restricted (IUGR) and appropriate for gestational age (AGA) groups.
hepatitis B, rheumatoid arthritis, renal insufficiency, asthma and psoriasis) in the remaining cases; all of the above resulted in small and infarcted placentas. Five mothers reported smoking 10 cigarettes per day. In the group of AGA infants, mothers were healthy; one smoked up to 5 cigarettes per day and another reported daily consumption of one glass of beer. All placentas in the AGA group were normal in appearance and weight. The neonates included in the study were single and did not present congenital infections or genetic syndromes. One- and fiveminute Apgar scores were in all IUGR cases and AGA controls N 7 and N 8, respectively. Demographic data of the participating infants, as well as of their mothers, are given in Table 1. Blood was drawn from the mothers at the first stage of labor or before receiving anesthesia in cases of elective cesarean section, from the doubly clamped umbilical cord. Blood was drawn from the neonates on days 1 (N1) and 4 (N4), characteristic of transition and stabilization to extrauterine life, respectively. Collected blood (240 samples in total) was centrifuged and supernatant plasma was kept frozen at − 80 °C until assay. The determination of all neurotrophins was performed by enzyme immunoassays [human β-NGF, Catalog Number DY256; human BDNF, Catalog Number DY248; human NT-3, Catalog Number DY267; human NT-4, Catalog Number DY268, (R&D Systems Inc, Minneapolis, MN 55413, USA)]. Minimum detectable concentration, intra- and inter-assay coefficients of variation were: for β-NGF 31.3 pg/mL, 8.9% and 12.7%, respectively; for BDNF 20 pg/mL, 7.5% and 11.3%, respectively; for NT-3 40.3 pg/mL, 8.2% and 11.9%, respectively; and for NT-4 25.8 pg/mL, 10.5%, and 15%, respectively.
3. Statistical analysis All neurotrophin data, except BDNF data, were not normally distributed (Kolmogorov–Smirnov test). Therefore, nonparametric procedures (Mann Whitney U test, Wilcoxon sum rank test, Spearman Rank correlation test) were applied in the statistical analysis as appropriate. P values b 0.05 were considered significant.
Amniotic fluid was diminished in all IUGR cases. IUGR and AGA fetuses did not present any differences in the umbilical cord artery pulsatility index (PI) and resistance index (RI), as demonstrated in Table 1. No statistically significant differences were observed among IUGR and AGA groups in maternal, fetal and neonatal day-1 and day-4 samples concerning levels of neurotrophins BDNF, NT-3 and NT-4. However, NGF levels were significantly higher in AGA maternal (p = 0.007), fetal (p = 0.01), and neonatal day-1 (p = 0.043) and day-4 (p = 0.003) samples as compared to respective levels in IUGR samples (Fig. 1). NGF levels in maternal, fetal and neonatal day-1 and day-4 samples positively correlated to the infants' centiles (p ranging from 0.04 to 0.0004) and birthweights (p ranging from 0.015 to 0.003). Nevertheless, BDNF, NT-3 and NT-4 levels neither in maternal, fetal or neonatal samples showed similar correlations with the infants' centiles and birthweights. Maternal levels were, in both IUGR and AGA groups, higher than respective fetal and neonatal levels (for all cases, p ranged from p b 0.001 to p = 0.04). Lastly, determined neurotrophin levels showed no correlation with gender or mode of delivery in both IUGR and AGA groups.
5. Discussion The results of this study indicate that no statistically significant differences were observed between fullterm IUGR and AGA groups concerning respective maternal, fetal and neonatal plasma levels of BDNF, NT-3 and NT-4. In contrast, NGF levels were significantly higher in all samples of the AGA group as compared to the IUGR group. During the perinatal period the blood–brain barrier is immature and, thus, circulating levels of neurotrophins may represent respective CNS levels [20]. Relatively, circulating BDNF levels have been previously shown to correlate with cortical BDNF levels [21]. Asymmetric IUGR cases are characterized by in utero hypoxia, which has been shown to influence a series of factors, mainly involved in angiogenesis, including angiopoietin-2 [22–24], vascular endothelial growth factor and placenta growth factor [25,26], endostatin [27,28] and soluble vascular endothelial growth factor receptor-1 [25,29]. Nevertheless, according to this study, in asymmetric fullterm IUGR infants probable intrauterine hypoxia does not seem to influence circulating – and presumably central – neurotrophin levels, a fact possibly implying the lack of CNS compromise, as neurotrophins are implicated in nervous system physiology. It is known that growth restriction successfully balances reduced oxygen delivery and consumption [30]. Thus, by restricting growth, the fetal organism manages to maintain or to minimally decrease arterial oxygen concentration, until substrate delivery is severely reduced [31]. However, minor reduction in overall oxygen consumption may mask large organ differences that, to some extent, mirror the pattern of blood flow redistribution [30]. As a consequence, cerebral metabolism is preserved, while oxygen consumption in
468 several organs, including the kidneys, is markedly decreased [30]. In reference to the latter, one can explain the diminished amniotic fluid [32], also observed in the present study. Although the mean customized centile of the asymmetric IUGR infants included in this study was considerably below the 10th customized centile, they did not present detectable changes in the umbilical and middle cerebral artery fetal Doppler studies and had an uncomplicated transition and stabilization to extrauterine life. In respect to this, it should be stressed that all infants included in the study reached term, thus growth restriction developed late in the third trimester, and therefore had a less adverse effect on the outcome than if it were initiated at an earlier stage of pregnancy, at a time when the brain might be more vulnerable [33] and preterm delivery would be unavoidable [34]. However, as already stated, all IUGR pregnancies were characterized by oligohydramnios, reflecting kidney hypoperfusion and blood redistribution (brain sparing effect) [18,12]. As for the deviating results of NGF, it has been shown that NGF levels measured in the umbilical cord of preterm infants were significantly lower than levels determined in fullterms [35]. In addition, we have also previously reported [36] that BDNF fetal and neonatal plasma concentrations were significantly higher in fullterm as compared to preterm cases. Although all IUGR infants included in this study were fullterms, one should take into consideration that they had a significantly lower birthweight than AGA infants, as prematures also have. Relatively, the present study showed that fetal and neonatal NGF plasma levels positively correlated with the centile and the birthweight of the infant, thus justifying higher NGF levels in the AGA group. It is noteworthy that this positive correlation did not apply to any other neurotrophin. On the other hand, Korhonen et al. [37] demonstrated that in infants with asphyxia, cerebrospinal fluid levels of NGF were significantly decreased as compared to the respective levels of infants without asphyxia. It could be speculated that similar findings are expected for NGF plasma levels, also in cases of in utero hypoxia. Although the umbilical artery pH values were statistically lower in the IUGR group compared to the AGA group in our study, they were not clinically significant, as all umbilical artery pH values were within normal limits. Furthermore, our study showed that in both IUGR and AGA groups, fetal and neonatal neurotrophin levels are lower than the respective maternal levels. This fact, confirming previous reports (referring only to BDNF in normal pregnancies, [36]) could be interpreted by taking into account the more mature peripheral and central nervous system of adults, given the developmental appearance of neurotrophins in humans [35]. Finally, this study did not find any statistical difference in neurotrophin levels both in IUGR and AGA groups between girls and boys [19,36]. Therefore, we can assume that neurodevelopment was similar in both genders. In conclusion, in the perinatal period, circulating levels of neurotrophins BDNF, NT-3 and NT-4 do not differ in IUGR and AGA pregnancies. In contrast, circulating NGF levels are higher in the AGA group; furthermore, NGF is the only neurotrophin for which the circulating levels are found to correlate with the customized centiles and the birthweights of the infants. Maternal neurotrophin levels are higher than
A. Malamitsi-Puchner et al. fetal or neonatal ones. Lastly, neither gender nor mode of delivery influence circulating neurotrophin levels in both IUGR and AGA groups at term. Further studies should investigate circulating neurotrophin levels in IUGR preterm infants, as well as in those with pronounced cerebral insults, manifested in prenatal blood flow Doppler studies.
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