Ö. KIRMEMİŞ, M. M. TATLI, C.TAYMAN, C. KOCA, A. KARADAĞ, N. URAS, U. DİLMEN, H. KAFALI Turk J Med Sci 2011; 41 (5): 795-800 © TÜBİTAK Original Article E-mail: [email protected] doi:10.3906/sag-1007-948

Subclinical of infants with intrauterine growth retardation determined by increased serum S100B protein levels

Özlem KIRMEMİŞ1, Mustafa Mansur TATLI2, Cüneyt TAYMAN2, Cemile KOCA3, Ahmet KARADAĞ4, Nurdan URAS5, Uğur DİLMEN5, Hasan KAFALI6

Aim: To test the hypothesis that serum S100B levels could be useful in detecting neurological damage in infants with intrauterine growth retardation (IUGR). Materials and methods: Th e study group consisted of infants with IUGR and the control group consisted of age- matched healthy infants. S100B protein levels were measured aft er birth and compared between groups. Results: For this study, 43 infants with IUGR and 25 infants as a control group were recruited. Gender, gestational age, type of delivery, and maternal age of the groups were statistically insignifi cant, with the exception of the mean birth weights (2120 ± 450 g in the IUGR group and 3096 ± 570 g in the control group (P < 0.001), respectively). S100B protein levels of the IUGR infants (1.13 ± 0.54) were signifi cantly higher than those of the control group (0.45 ± 0.13) (P < 0.001). IUGR infants treated with antenatal steroids showed lower S100B levels than IUGR infants that did not receive antenatal steroid treatments (P < 0.05). Th e study group infants were divided into 2 groups, for growth retardation (GR) that was asymmetric (n = 15) and symmetric (n = 28). Th e asymmetric and symmetric GR infants’ S100B levels were 1.14 ± 0.47 pg/mL and 1.21 ± 0.34 pg/mL, respectively, and no signifi cant diff erences were found between the 2 groups in terms of S100B levels (P = 0.32). Conclusion: Th e results of this study favor the opinion that there is an existing intrauterine hypoxia causing hypoxic brain tissue damage in IUGR infants, even when followed up with modern obstetrical screening protocols. Measurements of S100B may be useful in the prediction of outcome in these infants.

Key words: S100B, IUGR, intrauterine growth retardation, intrauterine hypoxia

İntrauterin büyüme gerelikli infantların subklinik hipoksilerinin artmış serum S100B protein seviyeleri ile saptanması

Amaç: Bu prospektif çalışmada intrauterin gelişme geriliği (IUGR) olan infantlarda nörolojik hasarı tespit etmede S100B seviyelerinin faydalı olduğu hipotezi test edildi. Yöntem ve gereç: Çalışma grubu İUGR infantlardan ve kontrol grubu benzer yaştaki sağlıklı infantlardan oluşmaktaydı. S100B protein seviyeleri doğum sonrası ölçüldü ve 2 grup karşılaştırıldı. Bulgular: Kırk üç IUGR ve kontrol grubunda 25 infant çalışmaya alındı. Cinsiyet, gestasyonel yaş, doğum şekli ve grupların maternal yaşı arasında ortalama doğum ağırlığı (sırasıyla IUGR grubunda 2120 ± 450 g kontrol grubunda

Received: 08.07.2010 – Accepted: 16.12.2010 1 Department of , Faculty of Medicine, Fatih University, Ankara - TURKEY 2 Department of Neonatology, Faculty of Medicine, Fatih University, Ankara - TURKEY 3 Division of Biochemistry, Faculty of Medicine, Fatih University, Ankara - TURKEY 4 Division of Neonatology, Faculty of Medicine, İnonü University, Malatya - TURKEY 5 Division of Neonatology, Dr. Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara - TURKEY 6 Division of Women’s Health, Faculty of Medicine, Fatih University, Ankara - TURKEY Correspondence: Cüneyt TAYMAN, Department of Neonatology, Faculty of Medicine, Fatih University, Ankara - TURKEY E-mail: [email protected]

795 S100B protein in intrauterine growth retardation

3096 ± 570 g (P < 0,001)) haricinde istatistiksel farklılık yoktu. S100B protein seviyleri IUGR infantlarda (1,13 ± 0,54) kontrol grubundan (0,45 ± 0,13) anlamlı derecede daha yüksek bulundu (P < 0,001). S100B seviyelerinin antenatal steroid tedavisi alan IUGR infantlarda antenatal steroit tedavisi almayan İUGR infantlara göre daha düşük düzeyde olduğu gösterildi (P < 0,05). Çalışma grubundaki infantlar asimetrik (n = 15) ve simetrik (n = 28) gelişme geriliği olmak üzere 2 gruba ayrıldılar. Asimetrik ve simetrik gelişme geriliği olan infantların S100B seviyeleri sırasıyla 1,14 ± 0,47 pg/mL ve 1,21 ± 0,34 pg/mL idi. İki grup arasında S100B seviyesi bakımından anlamlı farklılık bulunmadı (P = 0,32). Sonuç: Bu çalışmanın sonuçları modern obstetrik metodlarla takip edilse de, İUGR infantlarda hipoksik beyin hasarına neden olan intrauterin hipoksi varlığı düşüncesini desteklemektedir. Bu proteinin ölçülmesi bu infantların akibetlerini tahmin etmede yaralı olabilir.

Anahtar sözcükler: S100B, IUGR, intrauterin gelişme geriliği, intrauterin hipoksi

Introduction hypertonicity in their early life. In most situations, Placental insuffi ciency, with a consequent neurological examinations of IUGR infants are reduction in fetal nutritive and supply, is one uneventful and there is no evidence for the prediction of the most important causes of intrauterine growth of long-term neurological disorders in these babies restriction (IUGR) and fetal hypoxia, which is one of (3,4,6). Th erefore, the ability to monitor high-risk the main causes of and morbidity fetuses in the perinatal period by use of biochemical (1,2). Episodes of acute and chronic fetal hypoxia indexes could be particularly useful in detecting can result in brain lesions, which may leave long- cases at risk of adverse neurological outcomes, and term neurological sequelae such as auditory and in determining the timing of insults that damage visual impairment, mental retardation, and seizure the as early as possible with disorders. Moreover, minimal brain alterations, respect to future measures of prevention. such as a reduction in learning capabilities, dyslexia, S100B is an acidic calcium-binding protein and attention-defi cit/hyperactivity disorder, could that is released by brain tissue and is known to originate from the prenatal and/or perinatal exposure be highly specifi c to the nervous system (7). It is to hypoxia (2-4). Nowadays, the management thought to play a dual role in the regulation of cell of IUGR includes the combination of serial function, being benefi cial to cells at low doses but morphological ultrasound examinations to assess detrimental at high doses. Th e protein appears to be fetal growth and amniotic fl uid quantity, combined most abundant in glial cells, although its presence with functional testing such as a biophysical profi le, in neuronal subpopulations has also been reported. fetal heart rate testing, and Doppler examinations Th ese fi ndings have answered the question about the of fetal hemodynamics (5). Unfortunately, even if putative role of S100B in brain injury (8). Th erefore, hypoxia is detected, none of the presently available increased S100B concentrations in biological fl uids tests can defi ne the amount of hypoxia that the (e.g. cerebrospinal fl uid, blood, urine, and amniotic fetus can endure without developing hypoxic brain fl uid) of adults, infants, and fetuses aft er cell injury in lesions or the length of duration. Only the answers the nervous system have supported the use of S100B to these questions would enable the defi nition of the as a biochemical marker of brain damage (7). It was optimal timing of delivery in the case of chronic fetal found that this protein is increased in demyelinating hypoxia and prevention of perinatal brain damage. brain diseases, phenylketonuria, and especially Although in this situation the delay of the delivery in hypoxic-ischemic brain damage (HI) (7,9). might cause the prolongation of fetal exposure to S100B in the blood was measured, hypothesizing hypoxia, which could aff ect brain development and that during active brain injury, the protein could cause brain damage, early delivery carries the risk be released from the damaged tissue and part of of prematurity and associated risk of neurological it could spread into the systemic circulation, also disorders. Additionally, IUGR infants may show as a result of the hemodynamic rearrangement of hyperrefl exia, hyporefl exia, jitteriness, and/or the blood-brain barrier. In this regard, increased

796 Ö. KIRMEMİŞ, M. M. TATLI, C.TAYMAN, C. KOCA, A. KARADAĞ, N. URAS, U. DİLMEN, H. KAFALI

concentrations of S100B were detected 48-72 h from the umbilical vein for the standard assessments before any clinical, laboratory, or ultrasonographic (blood cell count, alanine aminotransferase, aspartate signs of intraventricular hemorrhage in preterm aminotransferase, blood urea, creatinine, glucose, newborns and of hypoxic ischemic encephalopathy electrolytes, interleukin-6, and C-reactive protein). (HIE) in full-term newborns (8). Currently, there are Infants that fulfi lled all of the following criteria few studies about S100B in the serum of infants with were admitted to the study: no maternal illness, no IUGR that are under risk of perinatal brain damage signs of fetal distress, a pH greater than 7.2 in cord (10-12). blood or venous blood, and Apgar scores above 7 In the present study, we aimed to investigate at 1 and 5 min. Exclusion criteria were multiple the serum S100B levels in infants with IUGR and maternal alcohol or cocaine to provide early prediction of possible negative addiction, infants with any malformation, cardiac neurodevelopmental outcomes, for both early and or renal diseases, hemolytic disease, or intrauterine later life in this particular population. infections. Peripheral venous blood samples for S100B Materials and methods measurement were obtained from a peripheral vein 12 h aft er birth and centrifuged immediately at 5000 Th e study was performed at 2 diff erent tertiary g for 10 min, and the sera were stored at –80 °C until referral centers for obstetrics and neonatal intensive concentration analyses were performed. All serum care units from February to July of 2007. We studied samples for S100B were of good quality without 43 women with singleton pregnancies complicated hemolysis. Th e S100B serum levels were determined by IUGR, between the 30th and 42nd weeks of by fully automatic electrochemoluminometric gestation. Informed consent was obtained from immunoassay (Elecsys S100®; Roche Diagnostics, both hospitals’ ethics committees and the parents. Penzberg, Germany) with a measurement range of Gestational age was determined by clinical data 0.005-39 ng/L. and by a fi rst trimester ultrasound scan. IUGR was defi ned by the presence of ultrasonographic signs A neurological examination was performed at (biparietal diameter below the 10th percentile and the same time as the blood sampling and during abdominal circumference below the 5th percentile) hospital discharge. Neonatal neurological conditions according to the nomograms of Campbell and were classifi ed as normal or abnormal. An infant Th oms (7,13). A drop in the percentile of fetal sizes was considered to be abnormal when one or more of was recorded between the fi rst scan aft er referral and the following neurological syndromes were present: the fi nal scan before delivery. Fetal growth restriction hypo- or hyperkinesias, hyper- or hypotonicity, was confi rmed by a birth weight below the 10th dystonia, hemisyndrome, apathy syndrome, or percentile in all of the fetuses. Th e ponderal index hyperexcitability syndrome. was calculated for each of the GR infants. Aft erwards, Statistical analysis infants were divided into 2 groups related to the For evaluation of the distribution of S100B ponderal index: asymmetric GR infants (<50% of measurement results, histogram and Kolmogorov- ponderal index) and symmetric GR infants (>50% Smirnov tests were applied. Student’s t-test was used of ponderal index) (14). Th e control group consisted for statistical evaluation of continuous variables. of 25 normal fetuses, matched for gestational age at Relationships between S100B protein levels and birth sampling (range: 30-42 weeks of gestation) and birth weight, birth length, maternal age, and gestational weights between the 10th and 90th percentiles. age were evaluated with Pearson’s correlation All women, in both the study and the control analysis. Th e gender of the infants, type of birth, groups, gave birth either vaginally or by elective and type of anesthesia were compared to each other cesarean section, based on obstetric indications. At using the chi-square test. Data were analyzed with delivery, the was clamped before any SPSS version 13.0 (SPSS Inc., Chicago, USA) with signs of breathing were seen and blood was drawn signifi cance defi ned as P < 0.05.

797 S100B protein in intrauterine growth retardation

Results dystonia, and hyperexcitability, were shown in 15 Th e fi ndings of the study are summarized in of the IUGR infants and in 11 of the control infants. the Table. Th e study group consisted of 43 infants Mean fetal S100B levels in the IUGR group (1.13 ± and the control group of 25 infants. No signifi cant 0.54 pg/mL) were signifi cantly higher (P < 0.001) than those in the control group (0.4 ± 0.13 pg/ diff erences between the 2 groups were found in terms mL). Th ere was a weak and low correlation between of gender, gestational age at delivery, type of delivery, gestational age and S100B protein levels (r = 0.24, P antenatal glucocorticoid treatment, and maternal = 0.12) and a weak and low correlation between birth age, with the exception of birth weight. Th e birth weight and S100B protein levels (r = –0.2, P = 0.006). weights of the 2 groups were 2120 ± 450 g in the When the IUGR fetuses were grouped according to IUGR group and 3096 ± 570 g in the control group antenatal steroid treatment, status of neurological (P < 0.001), respectively. Th ere were no signifi cant examination, type of delivery, and type of anesthesia diff erences in the laboratory parameters (red blood during delivery, those groups were found to be similar cell count, alanine aminotransferase, aspartate with respect to these parameters, except for the aminotransferase, blood urea, creatinine, glucose, presence of antenatal steroid treatment. IUGR infants electrolytes (sodium, potassium, and calcium), treated with antenatal steroids showed lower S100B interleukin-6, and C-reactive protein) between the levels than those without antenatal steroid treatment IUGR and control groups upon admission to the (P < 0.05). Th e study group infants were divided into neonatal intensive care unit (P > 0.005). Th e study 2 groups: asymmetric GR and symmetric GR. Th e and the control group were found to be similar asymmetric GR group consisted of 15 neonates, and with respect to neurological examinations. In both the symmetric SGA group consisted of 28 neonates. groups, none of the infants showed neurological Th e asymmetric and symmetric GR infants’ S100B abnormalities at the time of discharge from the levels were 1.14 ± 0.47 pg/mL and 1.21 ± 0.34 pg/ hospital, and no overt neurological syndromes were mL, respectively, and no signifi cant diff erences were observed during recovery. Isolated and transient found between the 2 groups in terms of S100B levels symptoms, including /, (P = 0.32).

Table. Characteristics of the study population and comparison of intrauterine growth retardation (IUGR) and control groups.

IUGR CONTROL P (n = 43) (n = 25)

Maternal age (mean ± SD, years) 28.4 ± 4.0 28.8 ± 4.5 0.7

Gestational age at delivery (mean ± SD, weeks) 37.7 ± 2.8 38.0 ± 2.5 0.6

Gender (male/female) 22/21 14/11 0.7

Birth weight (mean ± SD, g) 2120 ± 450 3096 ± 570 <0.001

Maternal glucocorticoid treatment (N/%) 11/25 5/20 0.5

Type of delivery (cesarean/vaginal) 26/17 13/12 0.5

Type of anesthesia during delivery (general/regional) 23/20 15/10 0.6

Neurological examination (normal/abnormal) 28/15 14/11 0.9

S100B protein (pg/mL) 1.13 ± 0.54 0.45 ± 0.13 <0.001

798 Ö. KIRMEMİŞ, M. M. TATLI, C.TAYMAN, C. KOCA, A. KARADAĞ, N. URAS, U. DİLMEN, H. KAFALI

Discussion in S100B levels between IUGR infants with normal Th e present study showed that S100B levels and abnormal short-term neurological outcomes increased in infants with IUGR at birth, and supports the concept that the neurological symptoms asymmetric and symmetric GR infants had similar observed in the fi rst few months aft er birth in S100B levels. Additionally, IUGR newborns with preterm infants who will develop an abnormal neurological outcome had similar are neither sensitive nor specifi c enough to ensure S100B levels as infants with IUGR and an uneventful reliable prognoses. Irritability, abnormal fi nger neurological follow-up. Th e present study was not posture, spontaneous Babinski refl ex, weakness of the fi rst to report the increased level of S100B protein the lower limbs, transient abnormality of tone, and a in IUGR infants. Previous human studies showed delay in achieving motor milestones are some of the that the continuous release of the protein from neurological signs that have been described in these damaged nervous tissue was the main cause of the high-risk preterm infants (6). All of these symptoms high level of S100B in blood obtained from preterm may be encountered before the onset of cerebral and term infants with IUGR, due to chronic hypoxia palsy or during “transient dystonia,” dissociated (7,10,12,15). Additionally, animal studies have shown motor development, and other transient neurological that intrauterine hypoxia is associated with persistent disturbances, which disappear during the fi rst or elevations of S100B protein in plasma, which supports second year of life. Moreover, no correlations have S100B as a useful early marker of fetal hypoxia (16). It been found among any of these symptoms and the was also shown that S100B protein levels in maternal severity of future motor impairment (6). Th erefore, and umbilical blood were increased in IUGR fetuses a traditional neurological examination fails to and represented cerebral cell damage in the perinatal predict the development and severity of long-term period (17). neurological abnormalities. Although S100B is a protein mainly concentrated Antenatal glucocorticoids are widely used in in the central nervous system, it was shown that premature infants, where they reduce neonatal pathological conditions that develop during morbidity and mortality. IUGR fetuses are at risk aff ect the S100B level in the , of preterm delivery and thus are likely to receive umbilical cord tissue, fetal membranes, amniotic antenatal glucocorticoids to promote lung maturation. fl uid, and the fetal cord blood (17). Th erefore, Because glucocorticoids alter vascular tone, it was biochemical markers in the umbilical vein blood questioned whether such treatment may induce also refl ect placental function. Th e half-life of this fetal cardiovascular alterations in IUGR infants; protein is approximately 1 h (4). Th erefore, placental some authors claimed that the fetal cardiovascular production might aff ect the serum levels of the response to maternal betamethasone administration protein in the early hours of life, but is obviously not in IUGR fetuses is signifi cantly diff erent than that responsible for the high levels found at later points seen in healthy fetuses (19). Th e IUGR fetus displays in time. It is suggested that elevated S100B levels 12 rebound cerebral perfusion that is not seen in the h aft er birth are a useful tool for the early detection healthy fetus, which is associated with increased of neurological events in infants (18). In this regard, oxidative stress and apoptosis in the fetal brain. in this study, blood samples were obtained from Th erefore, maternally administered glucocorticoids a peripheral vein 12 h aft er birth to measure the may have deleterious eff ects on the brain of IUGR accurate levels of S100B and to exclude placental fetuses. In light of this fact, it is logical to expect a contributions. higher level of S100B in steroid-administered groups, Th ese fi ndings, and the evidence that blood levels but our results contradicted this hypothesis rather of S100B were particularly high in IUGR newborns than supporting it. with and without abnormal short-term neurological To date, there have been no human studies that outcomes, together suggest that the level of S100B evaluated the relationship between antenatal steroid may refl ect the extent of brain damage aft er administration and S100B protein blood level in intrauterine hypoxic insult. Th e lack of diff erence IUGR infants. However, there was one animal study

799 S100B protein in intrauterine growth retardation

that investigated the eff ects of a single course of Conclusion antenatal betamethasone on S100B concentrations Th e results of this study and previous studies in preterm rats, in which a clinically equivalent dose warrant consideration and favor the opinion that and half of this dose was used (19). Th e authors there is an existing intrauterine hypoxia causing found that the hippocampal S100B content was hypoxic brain tissue damage in IUGR infants, even reduced by a clinically equivalent dose of 12 mg when followed up with modern obstetrical screening twice, but not by half of this dose. Interestingly, the protocols. Th e major goals of modern obstetric eff ect of betamethasone on brain cell proliferation screening procedures are the identifi cation of growth- also showed a dose-dependent pattern, as restricted fetuses among the small-for-gestational- investigated in the same hippocampal homogenates. age fetuses. Early detection of fetal hypoxia in high- Th e authors speculated that lowering the dose of risk pregnancies and timely termination of high-risk antenatal steroids may be less detrimental for brain pregnancies will prevent the development of hypoxic maturation. brain injury.

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