Mortality in Preterm Infants with Respiratory Distress Syndrome Treated with Poractant Alfa, Calfactant Or Beractant: a Retrospective Study

Journal of Perinatology (2013) 33, 119–125 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Mortality in preterm infants with respiratory distress syndrome treated with poractant alfa, calfactant or beractant: a retrospective study

R Ramanathan1, JJ Bhatia2, K Sekar3 and FR Ernst4 1LAC þ USC Medical Center and Children’s Hospital Los Angeles, Los Angeles, CA, USA; 2Division of Neonatology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA, USA; 3Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA and 4Premier Research Services, Premier healthcare alliance, Charlotte, NC, USA

infant deaths in 2005 and was the second leading cause of infant Objective: The objective of this study is to compare all-cause in-hospital mortality.2 Respiratory distress syndrome (RDS) is the most mortality in preterm infants with respiratory distress syndrome (RDS) common cause of respiratory distress in preterm infants and occurs treated with poractant alfa, calfactant or beractant. in nearly 50% of preterm infants born at less than 30 weeks of 3 Study Design: A retrospective cohort study of 14 173 preterm infants gestation. with RDS, treated with one of three surfactants between 2005 and 2009, Treatment with surfactant for RDS has been shown to using the Premier Database was done. Multilevel, multivariable logistic significantly decrease pneumothorax, and neonatal and infant 3–8 regression modeling, adjusting for patient- and hospital-level factors was mortality. The animal-derived surfactants, poractant alfa performed. (Curosurf, Chiesi Farmaceutici SpA, Parma, Italy), calfactant (Infasurf, Ony, St Louis, MO, USA) and beractant (Survanta, Abbott Result: Calfactant treatment was associated with a 49.6% greater Nutrition, Columbus, OH, USA) have been shown to be associated likelihood of death than poractant alfa (odds ratio (OR): 1.496, 95% with greater early improvement in the requirement for ventilatory confidence interval (CI): 1.014–2.209, P ¼ 0.043). Beractant treatment support, fewer pneumothoraces and reduced mortality when was associated with a non-significant 37% increase in mortality, compared with treatment with first-generation synthetic compared with poractant alfa (OR: 1.370, 95% CI: 0.996–1.885, surfactants.9,10 P ¼ 0.053). No differences in mortality were observed between calfactant Nine randomized, controlled clinical trials (RCTs)11–17 and one and beractant treatment (OR: 1.092, 95% CI: 0.765–1.559, P ¼ 0.626). retrospective study18 comparing these surfactant preparations in Conclusion: Poractant alfa treatment for RDS was associated with a the setting of RDS treatment have now been published. No significantly reduced likelihood of death when compared with calfactant significant differences in mortality were found in the four trials and a trend toward reduced mortality when compared with beractant. that compared beractant with calfactant.11,12 Among the five trials Journal of Perinatology (2013) 33, 119–125; doi:10.1038/jp.2011.125; that compared beractant with poractant alfa,13–16 one reported published online 1 September 2011 significantly lower mortality with poractant alfa in infants p32 weeks gestational age.15 Furthermore, in a meta-analysis of Keywords: surfactant; mortality; respiratory distress syndrome; poractant alfa; calfactant; beractant comparative trials, mortality was significantly lower (relative risk: 0.57, 95% CI: 0.34–0.96, P<0.05) with poractant alfa compared with beractant.3 In the only retrospective study published to date comparing surfactants, Clark et al.18 found no significant Introduction differences in all-cause mortality between beractant- and Preterm births continue to increase in spite of major advances in calfactant-treated patients overall or in any birth weight (BW) perinatal care, especially in developed countries.1 Prematurity and subgroups. low birth weight (LBW, < 2500 g) accounted for 16.5% of all There are no published studies comparing mortality in preterm infants treated with the three animal-derived surfactants available Correspondence: Dr FR Ernst, Premier Research Services, Premier healthcare alliance, 13034 in the US. RCTs using mortality as a primary outcome require Ballantyne Corporate Place, Charlotte, NC 28277, USA. large sample size, are expensive, and may take several years to E-mail: [email protected] Received 16 March 2011; revised 22 July 2011; accepted 25 July 2011; published online complete. The difficulties with conducting comparative RCTs in 1 September 2011 premature infants with RDS are evidenced by the premature Neonatal mortality with animal-derived surfactant use R Ramanathan et al 120 interruption of studies aimed at comparing different surfactant Statistical analysis preparations in terms of mortality or bronchopulmonary Patient demographics and hospital characteristics were compared dysplasia due to insufficient enrollment.12 To overcome these between treatment groups using the w2-test for categorical hurdles, we used data from a large national hospital variables. The comparison between surfactants in terms of database to assess whether there were differences in all-cause mortality was based on a mixed multilevel, multivariable logistic mortality among preterm infants treated with poractant alfa, regression model. The multilevel structure accounted for clustering calfactant or beractant. of infants within hospitals, including a random center effect in the model.21 Other than the type of surfactant, the following patient- Patients and methods level factors were included in the model to control for potentially A retrospective observational cohort analysis was conducted confounding variables: gestational age (categorized into 2-week using the US hospital administrative data from the Premier groups, from 25–26 weeks to 31–32 weeks), BW (categorized into Database.19–20 The Premier Database is a large US 250-g groups, from 500–749 g to 1750–1999 g), gender, race, 3M hospital-based database, containing information on approximately All Patient Refined Diagnosis Related Group severity of illness 22,23 5.5 million annual hospital discharges (approximately one- category and risk of mortality category. Furthermore, the fifth of all acute care hospitalizations in the US) with day-by-day following hospital-level factors were included as covariates: US service level detail. The Premier Data included hospitalizations Census region, population served (urban/rural), teaching status from more than 600 hospitals, approximately 30 of which (teaching/non-teaching) and hospital size (categorization based were children’s hospitals or had children’s hospital facilities. on the number of beds). These hospitals utilize the database for quality and To assess the sensitivity of the results, three alternative models utilization benchmarking. Hospitals submit data to the database, were estimated. In the first of these, gestational age at birth was which undergo quality checks and validation. As well, the data are excluded from the factors due to the potential inaccuracy in 24 also used by the US government agencies such as the Food gestational dating. In the second model, the covariates were and Drug Administration and Centers for Medicare and submitted to a backward selection procedure (removal from the Medicaid Services.19,20 model if P>0.1) to reduce the number of parameters and to obtain The analyses were conducted using de-identified data in more stable estimates of the effects of the surfactants. In the third compliance with the Health Insurance Portability and sensitivity model, the year during which the hospital discharge Accountability Act of 1996 (HIPAA). No institutional review board occurred was added as a covariate to account for potential trends in approval for the study was sought, as, in addition to being HIPAA mortality over time. All statistical analyses were performed using compliant, the de-identified nature of the database would preclude SAS 9.1.3 (SAS Institute Cary, NC, USA). the researchers from identifying any hospital sites or patients. The study was designed to compare all-cause in-hospital mortality, defined by the discharge status of ‘expired’, in preterm infants Results treated with poractant alfa, calfactant or beractant. A total of 14 173 infants discharged from 236 hospitals were included in the study population. Patient demographics and Study population hospital characteristics of the study population are shown in Infants were included in the study if they were discharged as an Table 1. inpatient from a Premier Database hospital from 1 January 2005, Overall, the unadjusted all-cause in-hospital mortality rates through 31 December 2009. Inclusion criteria for the study were 3.61% (n ¼ 184) in the poractant alfa group, 5.95% included having gestational age of 25–32 weeks, BW 500–1999 g, (n ¼ 201) in the calfactant group, and 4.58% (n ¼ 261) in the diagnosis of RDS, age p 2 calendar days, when they received the beractant group. When stratified by BW, as shown in Figure 1, the first dose of surfactant, and having received only one of the three lowest mortality rate was always observed in the poractant alfa study surfactants. group, except for the category 1250–1499 g, where beractant- Patients were excluded from the study in case of missing values treated infants had the lowest mortality. Mortality was significantly for any of the variables planned a priori to be included in the lower for infants 500–749 g, who received poractant alfa (11.72%) logistic regression model, if they received more than one surfactant than for those who received calfactant (20.67%, P<0.001) or during their hospitalization, or if there was evidence of congenital beractant (17.39%, P ¼ 0.011). In the 1000–1249 g BW category, abnormalities such as trisomy 13 or 18, anencephaly or dwarfism. mortality was significantly higher in the calfactant group (5.46%) Information on diagnosis of RDS, gestational age at birth, BW and than in the poractant alfa (2.67%, P ¼ 0.002) and beractant congenital anomalies was obtained from International (3.54%, P ¼ 0.035) groups. Classification of Diseases, 9th Revision, Clinical Modification The results of the multilevel, multivariable logistic regression (ICD-9) codes. model for all-cause in-hospital mortality are shown in Figure 2.

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Table 1 Patient demographics and hospital characteristics by surfactant treatment

Poractant alfa Calfactant Beractant All P vs C P vs B C vs B

n (%) n (%) n (%) n (%)

Discharges 5097 (100.0) 3378 (100.0) 5698 (100.0) 14 173 (100.0)

Calendar year of discharge <0.001 <0.001 <0.001 2005 653 (12.8) 832 (24.3) 1196 (21.0) 2681 (18.9) 2006 918 (18.0) 802 (23.7) 1363 (23.9) 3083 (21.8) 2007 1022 (20.1) 578 (17.1) 1135 (19.9) 2735 (19.3) 2008 1252 (24.6) 558 (16.5) 991 (17.4) 2801 (19.8) 2009 1252 (24.6) 608 (18.0) 1013 (17.8) 2873 (20.3)

Gestational age <0.001 <0.001 0.629 25–26 weeks 1016 (19.9) 754 (22.3) 1272 (22.3) 3042 (21.5) 27–28 weeks 1309 (25.7) 936 (27.7) 1544 (27.1) 3789 (26.7) 29–30 weeks 1427 (28.0) 909 (26.9) 1602 (28.1) 3938 (27.8) 31–32 weeks 1345 (26.4) 779 (23.1) 1280 (22.5) 3404 (24.0)

Birth weight 0.451 0.002 0.437 500–749 g 495 (9.7) 329 (9.7) 506 (8.9) 1330 (9.4) 750–999 g 1164 (22.8) 806 (23.9) 1419 (24.9) 3389 (23.9) 1000–1249 g 1160 (22.8) 770 (22.8) 1358 (23.8) 3288 (23.2) 1250–1499 g 959 (18.8) 664 (19.7) 1105 (19.4) 2728 (19.2) 1500–1749 g 806 (15.8) 498 (14.7) 822 (14.4) 2126 (15.0) 1750–1999 g 513 (10.1) 311 (9.2) 488 (8.6) 1312 (9.3)

Gender 0.475 0.489 0.179 Female 2308 (45.3) 1503 (44.5) 2618 (45.9) 6429 (45.4) Male 2789 (54.7) 1875 (55.5) 3080 (54.1) 7744 (54.6)

Race <0.001 <0.001 <0.001 White 2920 (57.3) 2185 (64.7) 2763 (48.5) 7868 (55.5) Black 1343 (26.3) 866 (25.6) 2100 (36.9) 4309 (30.4) Hispanic 564 (11.1) 251 (7.4) 698 (12.2) 1513 (10.7) Other 270 (5.3) 76 (2.2) 137 (2.4) 483 (3.4)

3M APR-DRG severity of illness <0.001 <0.001 <0.001 1 ¼ minor 47 (0.9) 33 (1.0) 46 (0.8) 126 (0.9) 2 ¼ moderate 561 (11.0) 268 (7.9) 418 (7.3) 1247 (8.8) 3 ¼ major 2436 (47.8) 1395 (41.3) 2670 (46.9) 6501 (45.9) 4 ¼ extreme 2053 (40.3) 1682 (49.8) 2564 (45.0) 6299 (44.4)

3M APR-DRG risk of mortality <0.001 0.008 <0.001 1 ¼ minor 1401 (27.5) 725 (21.5) 1422 (25.0) 3548 (25.0) 2 ¼ moderate 1907 (37.4) 1184 (35.1) 2128 (37.3) 5219 (36.8) 3 ¼ major 1472 (28.9) 1218 (36.1) 1783 (31.3) 4473 (31.6) 4 ¼ extreme 317 (6.2) 251 (7.4) 365 (6.4) 933 (6.6)

US census region of treating hospital <0.001 <0.001 <0.001 Northeast 371 (7.3) 648 (19.2) 450 (7.9) 1469 (10.4) Midwest 808 (15.9) 173 (5.1) 899 (15.8) 1880 (13.3) South 2715 (53.3) 2466 (73.0) 3988 (70.0) 9169 (64.7) West 1203 (23.6) 91 (2.7) 361 (6.3) 1655 (11.7)

Journal of Perinatology Neonatal mortality with animal-derived surfactant use R Ramanathan et al 122

Table 1 Continued

Poractant alfa Calfactant Beractant All P vs C P vs B C vs B

n (%) n (%) n (%) n (%)

Population served by treating hospital <0.001 <0.001 <0.001 Urban 4758 (93.3) 3313 (98.1) 5219 (91.6) 13290 (93.8) Rural 339 (6.7) 65 (1.9) 479 (8.4) 883 (6.2)

Teaching status of treating hospital <0.001 <0.001 <0.001 Teaching 3199 (62.8) 2281 (67.5) 3325 (58.4) 8805 (62.1) Non-teaching 1898 (37.2) 1097 (32.5) 2373 (41.6) 5368 (37.9)

Size (no. of beds) of treating hospital <0.001 <0.001 <0.001 <100 1 (0.02) 87 (2.6) 55 (1.0) 143 (1.0) 100–299 698 (13.7) 441 (13.1) 824 (14.5) 1963 (13.9) 300–499 2325 (45.6) 736 (21.8) 1760 (30.9) 4821 (34.0) 500+ 2073 (40.7) 2114 (62.6) 3059 (53.7) 7246 (51.1)

Abbreviations: 3M APR-DRG, 3M All Patient Reﬁned Diagnosis Related Group; B, beractant; C, calfactant; P, poractant alfa. P-values are based on the w2-test. Bold values are statistically signiﬁcant.

These results were supported by the sensitivity analyses performed. The increase in the likelihood of death with calfactant compared with poractant alfa was significant in the first two alternative models (51.9% increase, P ¼ 0.036 in the model excluding gestational age and 56.3%, P ¼ 0.016 in the backward selection model) and non-significant in the third model adding discharge year (35.0% increase, P ¼ 0.134). The trend towards an increased mortality with beractant compared with poractant alfa that was observed in the main model reached statistical significance in the first two alternative models (38.2% increase, P ¼ 0.048 and 37.7%, P ¼ 0.040, respectively), but did not reach significance in the third model (24.7% increase, P ¼ 0.179). In all alternative models, no differences in mortality were observed between calfactant and beractant. Figure 1 Unadjusted mortality rates by BW among the three surfactant-treated groups. Discussion The present study retrospectively investigated, for the first time, all- Calfactant was found to be associated with a 49.6% greater cause mortality among preterm infants with RDS, treated with the likelihood of death than poractant alfa (odds ratio (OR): 1.496, three animal-derived surfactants available in the US, namely, 95% confidence intervals (CI): 1.014–2.209, P ¼ 0.043). Beractant poractant alfa, calfactant or beractant. treatment was associated with a 37.0% increased mortality To overcome the difference in the demographic characteristics compared with poractant alfa, but the difference did not reach of the population investigated, which can be an intrinsic limitation statistical significance (OR: 1.370, 95% CI: 0.996–1.885, of retrospective studies, a logistic regression model adjusting for P ¼ 0.053). No differences in mortality were observed between patient and hospital factors was applied. Furthermore, the calfactant- and beractant-treated infants (OR: 1.092, 95% CI: clustering of infants within hospitals was accounted for by the 0.765–1.559, P ¼ 0.626). inclusion of the center effect in the model.

Journal of Perinatology Neonatal mortality with animal-derived surfactant use R Ramanathan et al 123

Odds Ratio (95% CI), p-value

Calfactant vs. Poractant alfa 1.496 (1.014 – 2.209), 0.043

Beractant vs. Poractant alfa 1.370 (0.996 – 1.885), 0.053

Calfactant vs. Beractant 1.092 (0.765 – 1.559), 0.626 • = Odds Ratio |— —| = 95% CI

0.0 0.5 1.0 1.5 2.0 2.5 3.0 Mortality Odds Ratio Figure 2 Comparison of mortality among the three surfactant-treated groups.

This model found calfactant to be associated with a significantly gestational age (n ¼ 270), a 3% mortality at 36 weeks post- greater likelihood of death than poractant alfa. Beractant was menstrual age in the poractant alfa (200 mg kg1)-treated infants associated with a non-significant increase in mortality, versus 11% for beractant (100 mg kg1)- or poractant alfa compared with poractant alfa, and no differences were observed (100 mg kg1)-treated patients (P ¼ 0.034 and P ¼ 0.046, between calfactant and beractant. The results obtained in respectively). In another RCT in 52 RDS patients, Fujii et al.17 the full model were also supported by the sensitivity analyses. reported that mortality was 8% in the poractant alfa 200 mg kg1 The alternative models showed a statistically significant group versus 19% in the beractant 100 mg kg1 group reduction of the likelihood death with poractant alfa compared (P ¼ 0.27). All together, these randomized, controlled studies with both calfactant and beractant, except the analysis including consistently showed a survival advantage with poractant alfa over discharge year, where the mortality reduction with poractant alfa beractant, although this reduction in mortality did not reach did not reach statistical significance. In particular, the model significance in most trials due to small sample size. Therefore, the which excluded gestational age was performed similar to the trend towards increased mortality with beractant compared approach followed by Clark et al.18 in the only retrospective with poractant alfa found in the present retrospective study comparison of calfactant and beractant published in the confirmed the findings of the smaller RCTs performed between literature, which used BW, but not gestational age, as a key these two surfactants. covariate. Some evidence suggests that, despite the fact that As far as beractant and calfactant comparisons, both RCTs and gestational age is a key factor in determining the outcome in retrospective evaluations have shown no mortality difference. The preterm infants, the methods to calculate it are not precise first RCT comparing beractant and calfactant in 1997 showed no unless an early first trimester fetal ultrasound was used for difference in mortality between these two surfactants in the overall estimating the gestational age.24 population.11 Additionally, Bloom et al.12 found 10% and 11% The unadjusted results were consistent with the adjusted model. mortality rates at 36 weeks post menstrual age for beractant- and Overall, the unadjusted mortality rates found in this study were calfactant-treated patients, respectively (pX0.05). Finally, in the 3.61% for poractant alfa, 4.58% for beractant and 5.95% for retrospective study by Clark et al.18 on 5169 infants, no differences calfactant. were found in mortality rates before 28 days of age between The results of this large retrospective study should be interpreted calfactant and beractant (OR: 1, 95% CI: 0.8–1.3). Our study and validated in the context of other evidence from the medical confirmed the absence of differences in mortality between beractant literature, which report comparisons between animal-derived and calfactant in prospective as well as retrospective studies surfactants. Focusing on poractant alfa and beractant, in a pilot published to date. study of 75 preterm infants with RDS by Speer et al.,13 mortality at Lastly, no study has been published comparing mortality 28 days was 3% in the poractant alfa 200 mg kg1 group and between poractant alfa and calfactant. This is therefore the first 12.5% in the beractant 100 mg kg1 group; however, this difference direct comparison available between these two surfactants, showing did not reach significance (adjusted OR: 0.23, 95% CI: 0.02–2.54, a significant greater likelihood of death with calfactant than P ¼ 0.23). In a prospective study of 58 RDS infants, Malloy et al.16 poractant alfa. found no significant difference in mortality at 40 weeks between Our study has certain limitations due to the retrospective nature infants receiving poractant alfa and beractant (0 vs 10%, of the database used. Among the restrictions of the database, respectively P ¼ 0.08). A larger study by Ramanathan et al.15 in information on the precise cause of death is unavailable and the 293 RDS infants, found in those who were no more than 32 weeks number of surfactant doses is not reliably calculable. The database

Journal of Perinatology Neonatal mortality with animal-derived surfactant use R Ramanathan et al 124 also lacked reliable antenatal steroid use data, partly because and interacting with surfactant protein B to regulate the adsorption antenatal steroids may have been given to the mother before entry and spreading properties of the phospholipids.30 into the hospital for delivery, and the Premier Database focused on In conclusion, this large retrospective study of preterm infants hospital data by design. It was not possible, therefore, to adjust the with RDS found lower mortality among infants who received model for this factor as a covariate, despite the importance that poractant alfa, compared with infants who received either antenatal steroid use has for improving lung function and calfactant or beractant, even after adjusting for patient reducing RDS severity and its related mortality risk. However, in the characteristics such as gestational age and BW, and after Clark et al.18 study where data on antenatal steroids were available accounting for hospital characteristics and center effects. These and the comparison between surfactants was adjusted for this results in real-world settings are consistent with prior RCTs, but covariate, the finding of no difference in the outcome between provide additional significant findings that most RCTs have not calfactant and beractant was coherent with our study results. been able to provide due to their relatively small sample size. We acknowledge that the value of retrospective studies, despite their limitations, lies in the possibility to study large patient sample size, contributing to increased study power. This is particularly Conflict of interest important in the field of clinical investigation on surfactants, This study was sponsored by Chiesi Farmaceutici SpA, the where the known efficacy of treatment on mortality outcomes manufacturer of poractant alfa. Frank R Ernst is an employee of implies the need for large sample size to detect even small, but Premier, which contracted with Chiesi Farmaceutici SpA to conduct significant difference, making RCTs often unaffordable in terms of the study. Rangasamy Ramanathan, Kris Sekar and Jatinder Bhatia costs and recruitment. As an example, the treatment trial published have served as consultants to Chiesi Farmaceutici SpA. by Bloom et al.12 required a sample size of 2080 infants to detect a 6% difference in infants alive without bronchopulmonary dysplasia between calfactant and beractant. However, the study was Acknowledgments terminated prematurely after enrollment of 1361 infants (65.4% of the target). Finally, emerging evidence shows that findings from We would like to thank Stefano Vezzoli for his extensive statistical support. The retrospective studies may provide the medical community with authors also thank Raffaella Monno and Carmen Dell’Anna for their scientific contribution and assistance with the manuscript, as well as Teresa Davis for her information on drug effectiveness in real-world settings.25 database programming support. The lower mortality observed in poractant alfa-treated infants compared with calfactant or beractant prompts one to look for a possible explanation for such different outcomes for poractant alfa References over the other two surfactants. The most likely explanation may be due to different surfactant doses administered to the infants 1 Martin JA, Kung HC, Mathews TJ, Hoyert DL, Strobino DM, Guyer B et al. Annual included in the database, according to their US-prescribing summary of vital statistics: 2006. Pediatrics 2008; 121(4): 788–801. 1 1 2 Kung HC, Hoyert DL, Xu JQ, Murphy SL. Deaths: Final data for 2005. National Vital information: 200 mg kg for poractant alfa, 100 mg kg for Statistics Reports. 56(10). National Center for Health Statistics: Hyattsville, MD. 1 beractant and 105 mg kg for calfactant. Poractant alfa is the http://www.cdc.gov/nchs/data/nvsr/nvsr56/nvsr56_10.pdf, 2008 (accessed on only surfactant that has been studied using 200 mg kg1 for the 21 December 2010). initial dose, and has been associated with faster weaning of oxygen 3 Halliday HL. History of surfactant from 1980. Biol Neonate 2005; 87: 317–322. and peak inspiratory pressure, fewer doses and lower mortality. 4 Whitsett JA, Weaver TE. Hydrophobic surfactant proteins in lung function and disease. 1 N Engl J Med 2002; 347: 2141–2148. Evidence from a RCT has shown that poractant alfa 200 mg kg 5 Ballard PL, Merrill JD, Godinez MH, Troug WE, Ballard RA. Surfactant protein profile 1 is better than poractant alfa 100 mg kg in reducing mortality, of pulmonary surfactant in premature infants. Am J Respir Crit Care Med 2003; 168: whereas when poractant alfa and beractant are used at the same 1123–1128. dose of 100 mg kg1 for the initial dose, no difference in mortality 6 Minoo P, Segura L, Coalson JJ, King RJ, DeLemos RA. Alterations in surfactant protein was observed, despite the faster onset of action with poractant gene expression associated with premature birth and exposure to hyperoxia. Am J alfa.15 Compared with poractant alfa 100 mg kg1, poractant alfa Physiol 1991; 261: L386–L392. 1 7 Merrill JD, Ballard RA, Cnaan A, Hibbs AM, Godinez RI, Godinez MH et al. Dysfunction 200 mg kg has also been shown to result in longer surfactant of pulmonary surfactant in chronically ventilated premature infants. Pediatr Res 2004; 26 half-life, fewer retreatments and improved oxygenation. Poractant 56: 918–926. alfa is the surfactant preparation that closely resembles 8 Horbar JD, Wright EC, Onstad L, Members of the National Institute of Child health and phosphatidylcholine molecular species composition of human Human Development Neonatal Research Network. Decreasing mortality associated with surfactant and contains27,28 the highest amount of polyunsaturated the introduction of surfactant therapy: an observational study of neonates weighing 601–1300 grams at birth. Pediatrics 1993; 92: 191–196. fatty acid-phospholipids and plasmalogens amongst other 9 Soll RF, Blanco F. Natural surfactant extract versus synthetic surfactant for neonatal surfactant preparations, when normalized for phospholipid respiratory distress syndrome. Cochrane Database Syst Rev 2001; 2: CD0014444. 29 amounts. These components are important for reducing viscosity 10 Halliday HL. Surfactants: past, present and future. J Perinatol 2008; 28: S47–S56.

Journal of Perinatology Neonatal mortality with animal-derived surfactant use R Ramanathan et al 125

11 Bloom BT, Kattwinkel J, Hall RT, Delmore PM, Egan EA, Trout JR et al. 22 Averill R, Goldfield N, Hughes J, Muldoon J, Gay J, Mc Cullough E et al. What are Comparison of Infasurf (calf lung surfactant extract) to Survanta (Beractant) in APR-DRGs? An introduction to severity of illness and risk of mortality adjustment the treatment and prevention of respiratory distress syndrome. Pediatrics 1997; 100: methodology (White Paper). 3M Health Information Systems: Saltlake City, UT, 2003, 31–38. http://solutions.3m.com/3MContentRetrievalAPI/BlobServlet?locale ¼ it_IT&lmd ¼ 12 Bloom BT, Clark RH. Comparison of Infasurf (calfactant) and Survanta (beractant) in 1218718280000&assetId ¼ 1180603360910&assetType ¼ MMM_Image&blob- the prevention and treatment of respiratory distress syndrome. Pediatrics 2005; 116: Attribute ¼ ImageFile (accessed on 10 February 2011). 392–399. 23 Muldoon JH. Structure and performance of different DRG classification systems for 13 Speer CP, Gefeller O, Groneck P, Laufko¨tter E, Roll C, Hanssler L et al. neonatal medicine. Pediatrics 1999; 103(1 Suppl E): 302–318. Randomised clinical trial of two treatment regimens of natural surfactant preparations 24 Savitz DA, Terry Jr JW, Dole N, Thorp Jr JM, Siega-Riz AM, Herring AH. Comparison of in neonatal respiratory distress syndrome. Arch Dis Child Fetal Neonatal Ed 1995; 72: pregnancy dating by last menstrual period, ultrasound scanning, and their F8–13. combination. Am J Obstet Gynecol 2002; 187: 1660–1666. 14 Baroutis G, Kaleyias J, Liarou T, Papathoma E, Hatzistamatiou Z, Costalos C. 25 Iglehart JK. Prioritizing comparative-effectiveness researchFIOM recommendations. Comparison of three treatment regimens of natural surfactant preparations in neonatal N Engl J Med 2009; 361: 325–328. respiratory distress syndrome. Eur J Pediatr 2003; 162: 476–480. 26 Cogo PE, Facco M, Simonato M, Verlato G, Rondina C, Baritussio A et al. Dosing of 15 Ramanathan R, Rasmussen MR, Gerstmann DR, Finer N, Sekar K, North American porcine surfactant: effect on kinetics and gas exchange in respiratory distress syndrome. Study Group. A randomized, multicenter masked comparison trial of poractant alfa Pediatrics 2009; 124: e950–e957. (curosurf) versus beractant (survanta) in the treatment of respiratory distress syndrome 27 Bernhard W, Mottaghian J, Gebert A, Gunnar AR, von der Hardt H, Poets CF. in preterm infants. Am J Perinatol 2004; 21: 109–119. Commercial versus native surfactants. Surface activity, molecular components, and the 16 Malloy CA, Nicoski P, Muraskas JK. A randomized trial comparing beractant and effect of calcium. Am J Respir Crit Care Med 2000; 162: 1524–1533. poractant treatment in neonatal respiratory distress syndrome. Acta Paediatr 2005; 94: 28 Blanco O, Pe´rez-Gil J. Biochemical and pharmacological differences between 779–784. preparations of exogenous natural surfactant used to treat respiratory distress 17 Fujii AM, Patel SM, Allen R, Doros G, Guo CY, Testa S. Poractant alfa and beractant syndrome: role of the different components in an efficient pulmonary surfactant. Eur J treatment of very premature infants with respiratory distress syndrome. J Perinatol. Pharmacol 2007; 568: 1–15. 2010; 30: 665–670. 29 Ru¨diger M, To¨lle A, Meier W, Ru¨stow B. Naturally derived commercial surfactants differ 18 Clark RH, Auten RL, Peabody J. A comparison of the outcomes of neonates treated with in composition of surfactant lipids and in surface viscosity. Am J Physiol Lung Cell Mol two different natural surfactants. J Pediatr 2001; 139: 828–831. Physiol 2005; 288: L379–L383. 19 Premier Database. Premier healthcare alliance. Premier: Charlotte, NC. 30 To¨lle A, Meier W, Greune G, Ru¨diger M, Hofmann P, Ru¨stow B. Plasmalogens reduce http://premierinc.com/quality-safety/tools-services/prs/data/perspective.jsp the viscosity of a surfactant-like phospholipid monolayer. Chem Phys Lipids 1999; 100: (accessed on 12 December 2010). 81–87. 20 Premier Research Services. Premier healthcare alliance. Bibliography of Peer-Reviewed publications: Charlotte, NC. http://www.premierinc.com/quality-safety/ This work is licensed under the Creative tools-services/prs/published-research/index.jsp (accessed on 10 February 2011). Commons Attribution-NonCommercial-No 21 Clarke P. When can group level clustering be ignored? Multilevel models versus Derivative Works 3.0 Unported License. Toview a copy of single-level models with sparse data. J Epidemiol Community Health 2008; 62: this license, visit http://creativecommons.org/licenses/ 752–758. by-nc-nd/3.0/

Journal of Perinatology Journal of Perinatology (2013) 33, 161–167 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp LETTERS TO THE EDITOR Is there evidence for a mortality difference between natural surfactants?

Journal of Perinatology (2013) 33, 161–162; doi:10.1038/jp.2011.196; There are significant pitfalls when using an administrative published online 16 February 2012 database to conduct clinical research that the authors do not adequately address. Although they did correctly note that retrospective analyses are subject to significant bias and error, they I read with interest the recent paper by Ramanathan et al.,1 failed to take steps often found in retrospective studies to reduce that compared outcomes among neonates treated with different these risks. Specifically, there was no attempt to obtain missing animal-derived surfactants. The authors’ conclusion that poractant data or even verify the data that were included. alfa may offer a survival advantage over calfactant or beractant Although the authors cited several articles that they believed contradicts everything we understand about the biology of natural are consistent with their findings, these are primarily limited surfactants, and virtually all prospective clinical data we have to those studies showing no clinical differences between calfactant gathered for the past 30 years. They base their conclusion on a and beractant. Although they stated in their paper that the retrospective review using a limited administrative data set, an results of their study ‘should be interpreted and validated in approach with inherent weaknesses that makes such a paradigm- the context of other evidence from the medical literature’, shifting claim untenable. they were unable to do so because there is simply no literature Their claim is not entirely new. The authors have previously supporting a mortality difference between natural surfactants, published abstracts of portions of this data set, and presented their as noted in a previous review5 (that the authors also failed to cite findings at a variety of forums since 2007. What is particularly in their paper). Moreover, the authors omitted reference to the concerning is that in the present article the authors have omitted largest retrospective study to date comparing outcomes by natural important information previously presented that would underscore surfactant preparation that failed to show any differences in some of the weaknesses of the present study. Specifically, mortality.4 (1) In a previous abstract that reported from the same data source The authors’ speculation about the reason for outcome from January 2003 through June 2006 (overlapping the differences between surfactants also conflicts with the body of present report’s data set by 2 years) the authors note that evidence from animal and clinical studies. They suggest that nearly 60% of the cases were omitted due to incomplete data.2 different doses of phospholipid may explain differences in outcome, Omitting such a large number of cases precludes any reliable although the phospholipid content in all commercially available conclusions. In the present study the authors do not state products far exceeds the lung’s normal phospholipid pool size,6 how many cases were omitted. and has not been demonstrated to affect outcome in large (2) In a previous but separate abstract that reported from the randomized clinical trials. Instead, the authors cited their own same data source as above, the authors found that neonatal previous small trial in which a post hoc subgroup analysis intensive care unit and hospital lengths of stay (LOS) were suggested that a 200 mg kg1 dose of poractant alfa was associated significantly lower in poractant alfa-treated infants than in with lower mortality than 100 mg kg1 doses of either poractant either beractant- or calfactant-treated infants.3 Shorter LOS alfa or beractant in preterm infants at 36 weeks postconceptional coupled with lower mortality suggests that poractant alfa- age (P ¼ 0.05).7 This finding is inconsistent with all other treated infants were significantly more mature, less ill or both. outcomes reported in their study, including survival at 28 days and This is consistent with a large retrospective database analysis survival without chronic lung disease, which did not differ among published earlier this year4 (although not cited by the authors groups in either the overall or the subgroup analyses. It also in the present study). conflicts with data from an earlier randomized prospective trial (3) In the present study, the authors do not report LOS and do not that included >10-fold greater numbers of poractant-treated case-adjust for LOS in any of their analyses. Although they infants, and which found no differences in any clinically important attempt to adjust for case mix and mortality risk by All Patient outcomes, including mortality, between infants treated with Refined Diagnosis Related Groups this approach has not been 200 mg kg1 poractant alfa and infants treated with 100 mg kg1.8 validated for this type of study, particularly when a large However, the authors did not discuss this important study percentage of the cases are omitted due to incomplete data. in their present report. Letters to the Editor 162

Given the inherent weaknesses in this type of retrospective study, References the omission of a significant number of cases as well as important 1 Ramanathan R, Bhatia JJ, Sekar K, Ernst FR. Mortality in preterm infants with covariates in the analysis, and the weight of previously published respiratory distress syndrome treated with poractant alfa, calfactant, or beractant: a data relevant to this important clinical question, the authors retrospective study. J Perinatol 2013; 33: 119–125. should exert extreme caution in interpreting their findings. In a 2 Bhatia J, Saunders WB, Friedlich P, Lavin PT, Sekar KC, York JM et al. Differences in recent review on this precise topic, I concluded ‘there is no valid mortality among infants treated with three different natural surfactants for respiratory distress syndrome. E-PAS 2007; 617935.16 (abstract). evidence for a mortality benefit of one surfactant preparation over 3 Sekar KC, Bhatia J, Ernst FR, Saunders WB, Lavin PT, Ramanathan R. Resource use in 5 another’. This study by Ramanathan et al. has not altered my preterm neonates with respiratory distress syndrome (RDS) treated with one of three assessment. difference natural surfactants: analyses using a large hospital discharge database. Acta Paediatr 2007; 96(Suppl 456): 109 (abstract). 4 Trembath AN, Clark RH, Bloom BT, Smith PB, Bose C, Laughon M. Trends in surfactant use Conflict of interest in the United States: changes in clinical practice. E-Journal Neo Res 2011; 1(1): 23–30. 5 Holm B, Cummings JJ. Is there evidence for a mortality difference between exogenous Dr Cummings has served as a consultant for ONY, surfactant preparations in neonatal RDS? J Appl Res 2008; 8(2): 78–83. manufacturer of calfactant, and for Discovery Labs, manufacturer 6 Carnielli VP, Zimmermann LJ, Hamvas A, Cogo PE. Pulmonary surfactant kinetics of the of lucinactant. He currently has no financial interests with any newborn infant: novel insights from studies with stable isotopes. J Perinatol 2009; 29: surfactant manufacturer. S29–S37. 7 Ramanathan R, Rasmussen MR, Gerstmann DR, Finer N, Sekar K, North American Study Group. A randomized, multicenter, masked comparison trial of poractant alfa (Curosurf) versus beractant (Survanta) in the treatment of respiratory distress syndrome JJ Cummings in preterm infants. Am J Perinatol 2004; 21(3): 109–119. Pediatrics and Physiology, East Carolina University, 8 Halliday HL, Tarnow-Mordi WO, Corcoran JD, Patterson CC. Multicentre randomized The Brody School of Medicine, Greenville, NC, USA trial comparing high and low dose surfactant regimens for the treatment of respiratory E-mail: [email protected] distress syndrome (the Curosurf 4 trial). Arch Dis Child 1993; 69: 276–280.

Response to Cummings

Journal of Perinatology (2013) 33, 162–165; doi:10.1038/jp.2011.197; and dose of the surfactant. PA was bactericidal in a dose-dependent published online 16 February 2012 fashion and differed from BE and BO, a surfactant preparation similar to CA.14 PA contains the highest amount of plasmalogens (PL) when compared with BE.15 BO contains the lowest amount of We would like to thank Dr Cummings for giving us the opportunity PL.15 PL are anti-oxidant phospholipids and presence of higher to explain our study results. Results from nine randomized, amounts of PL in the tracheal aspirates from pre-term infants has controlled, trials (RCTs),1–9 including the one that has been been shown to be associated with a lower risk for bronchopulmonary just published (e-pub ahead of print) by Dizdar et al.9 and dysplasia (BPD).16 Also, the amount of surfactant-associated meta-analyses10–12 comparing animal-derived surfactants, namely, protein B (SP-B) is highest in PA when compared with BE or CA. poractant alfa (PA), beractant (BE), bovactant (BO) and/or SP-B is the most important SP in helping the phospholipids to calfactant (CA), have consistently shown faster weaning of rapidly adsorb at the air–liquid interphase and in decreasing oxygen and mean airway pressure, less need for redosing, fewer surface tension. Furthermore, the phospholipid molecular species days on oxygen and mechanical ventilation, shorter length of stay of PA is much closer to that of human surfactant.17,18 (LOS) as well as survival advantage in babies treated with PA. In our paper,19 we have clearly acknowledged the limitations These advantages with PA over BE, CA or BO are likely related to of the retrospective study and took steps to minimize any major biological and/or biochemical differences between these shortcomings. The major finding of lower mortality in PA-treated animal-derived surfactant preparations. PA contains the highest infants is consistent with results from previously published small amount of phospholipids when compared with BE or CA. Higher RCTs as well as meta-analyses comparing PA and BE. None of the amount of phospholipids has been shown to downregulate oxidative studies comparing BE with CA have shown any differences in the functions in monocytes and confer better anti-inflammatory need for redosing, days on oxygen or mechanical ventilation, BPD properties.13 In addition, bacterial growth in different surfactant or mortality. Interestingly, Bloom et al.1 reported a higher preparations is influenced by microbial species and the composition mortality rate in infants with a birth weight <600 g treated with CA

Journal of Perinatology Letters to the Editor 162

Response to Cummings

Journal of Perinatology Letters to the Editor 163 as compared with BE (63% vs 26%, P<0.007). However, this those treated with BE (weighted mean difference: 26.3 fewer finding was not duplicated in a retrospective study using a days (95% CI: 36.6 to 16.1); P<0.00001). Additionally, it is database, similar to our study, published by Clark et al.20 not generally considered appropriate to adjust the results of In regards to the concern regarding results of the PA versus one outcome measure (for example, mortality) for another BE RCT published in 2004, Ramanathan et al.6 had clearly stated, outcome measure (LOS). Adjustments for case mix and a priori, under the methods section that they would evaluate mortality risk by APR-DRGs are very commonly done in ‘oxygen requirement at 36 weeks post-conceptional age for infants studies involving large databases, such as the one that was born at p32 weeks gestation, and mortality’ as secondary used in our study. The appropriateness of APR-DRG’s method outcomes. Therefore, this was not a post hoc analysis as stated in in terms of structure and statistical performance in neonatal the review article by Holm and Cummings.21 medicine has been extensively discussed by Muldoon.24 Responses to specific comments Moreover, only 69 patients in our study lacked APR-DRG information, so severity of illness was accounted for in our (1) A different study, published as an abstract in 2007, and using study. In addition, the data on maturity (gestational age the Premier database, applied a different time frame, patient and birth weight) presented in our paper and accounted for population and statistical approach, and reached similar in our mortality regression modeling, help rule out differences conclusions, namely survival advantage with PA when in maturity as a substantial explanation for the differences compared with BE or CA.22 In the 2007 abstract, the authors in mortality results. noted that 10 237 of the 24 907 patients meeting basic study criteria were included in the adjusted analysis resulting from As stated previously, there have been no significant differences in the regression model; this was deemed acceptable, as unknown the three RCTs comparing CA with BE.1–3 In the review article by (unreported, missing or invalid) values of the covariates do Holm and Cummings,21 they did not state the mortality differences not permit appropriate adjustment for case mix. In the current between PA and BE from RCTs. study, there were 8276 patients who met the selection criteria, Surfactant dosing has clearly been shown to affect the clinical yet were excluded due to unreported, missing or invalid entries outcomes. Following is the discussion by Singh et al.12 from the for one or more of the variables: gender, race, APR-DRG, most recent meta-analysis: gestational age or birth weight. The exclusion of these left Despite these limitations, this meta-analysis differs from other 14 173 patients for use in the revised regression models, which reviews in that it includes the largest number of RCTs comparing now also accounted for center effects. Furthermore, the poractant alfa and beractant, with a fairly large cumulative sample outcome that was analyzed in this paper was limited to size. The superior outcomes noted in this meta-analysis with mortality only. poractant alfa, compared with beractant, particularly with respect (2) The lower mortality observed with PA treatment cannot be to reductions in mortality rates, the need for redosing and initial attributed to the idea that a less ill or more mature population respiratory support, might be attributable to differences in the received this treatment. In the analysis stratified by birth biochemical and biophysical properties of poractant alfa itself or to weight, the lowest mortality rate was always observed in the PA the initial higher dose (200 mg kg1) used. The higher dose of group, except for the category 1250–1499 g. In fact, our study poractant alfa, with greater contents of phospholipids and results of decreased mortality coupled with fewer days on surfactant-associated proteins B and C, might have resulted oxygen and mechanical ventilation has been reported in a in greater improvements in lung function and hence better recent meta-analysis comparing porcine versus bovine-derived outcomes. Because the low dose of poractant alfa (100 mg kg1) surfactants.12 We did not cite the report by Trembath et al.23 in did not have the same effects on mortality and redosing rates which they described trends in surfactant use due to the clearly as did the high dose of poractant alfa, it seems that the greater different objective (no pre-specified analysis on mortality) and concentrations of phospholipids and surfactant proteins in population (more mature infants with no specified RDS a given volume might be responsible for the observed diagnosis) compared with our study. In the population superior effects when high-dose poractant alfa is compared considered by Trembath et al.,23 it may be difficult to with beractant at the dosage volumes recommended by the demonstrate significant differences between treatments in terms manufacturers. of mortality due to the limited number of deaths observed. In the five RCTs comparing PA 200 mg kg1 with BE (3) The analysis reported in this paper19 did not have the aim 100 mg kg1, faster weaning of oxygen and peak inspiratory of evaluating differences between surfactants in terms of LOS. pressure, less need for redosing, fewer cases of air leaks and In the recent meta-analysis12 comparing porcine versus clinically significant patent ductus arteriosus and earlier bovine-derived surfactants, the length of hospital stay was extubations in PA-treated infants have been reported.4,6–9 These significantly shorter for infants treated with PA, compared with acute benefits in PA-treated infants could potentially result in

Journal of Perinatology Letters to the Editor 164 shorter LOS. In fact, in the most recent RCT, Dizdar et al.9 reported References significantly more infants surviving without BPD (78.7 vs 58.5%, 1 Bloom BT, Kattwinkel J, Hall RT, Paula M, Delmore PM, Egan EA et al. Comparison P ¼ 0.015) without increase in LOS in the PA-treated group versus of Infasurf (calf lung surfactant extract) to Survanta (Beractant) in the treatment BE group. There was also a trend towards fewer deaths in PA- vs and prevention of respiratory distress syndrome. Pediatrics 1997; 100: 31–38. BE-treated patients (9.8 vs 20%). In a meta-analysis by Fox and 2 Attar MA, Becker MA, Dechert RE, Donn SM. Immediate changes in lung compliance Sothinathan,10 PA-treated infants were less likely to need more following natural surfactant administration in premature infants with respiratory distress syndrome: a controlled trial. J Perinatol 2004; 24: 626–630. than one dose than those treated with BE (37 vs 53%; RR 1.36; 95% 3 Bloom BT, Clark RH; Infasurf Survanta Clinical Trial Group. Comparison of Infasurf CI 1.08, 1.72). In a multicenter RCT, comparing high versus low- (calfactant) and Survanta (beractant) in the prevention and treatment of respiratory 25 dose bovine surfactant, Surfactant-TA, Konishi et al. showed distress syndrome. Pediatrics 2005; 116: 392–399. significantly less intraventricular hemorrhage and BPD in pre-term 4 Speer CP, Gefeller O, Groneck P, Laufko¨tter E, Roll C, Hanssler L et al. Randomised infants treated with the high dose. In a study comparing high- clinical trial of two treatment regimens of natural surfactant preparations in versus low-dose BO, Gortner et al.26 reported that high-dose BO neonatal respiratory distress syndrome. Arch Dis Child Fetal Neonatal Ed 1995; 72: 8–13. improved oxygenation and reduced barotrauma. In a study of 5 Baroutis G, Kaleyias J, Liarou T, Papathoma E, Hatzistamatiou Z, Costalos C. 27 single versus multiple doses of PA by Speer et al. multiple dose Comparison of three treatment regimens of natural surfactant preparations in neonatal treatment resulted in a reduced incidence of pneumothorax (18 vs respiratory distress syndrome. Eur J Pediatr 2003; 162: 476–480. 9%, P<0.001) and more importantly, a reduction in mortality 6 Ramanathan R, Rasmussen MR, Gerstmann DR, Finer N, Sekar K, North American (21 vs 13%, P<0.05). Our results do not conflict with data from Study Group. A randomized, multicenter masked comparison trial of poractant alfa (curosurf) versus beractant (survanta) in the treatment of respiratory distress syndrome an earlier randomized prospective trial that included more than in preterm infants. Am J Perinatol 2004; 21: 109–119. 28 10-fold greater number of PA-treated infants. We did not discuss 7 Malloy CA, Nicoski P, Muraskas JK. A randomized trial comparing beractant and this study because the ‘low-dose’ PA-treated group could receive a poractant treatment in neonatal respiratory distress syndrome. Acta Paediatr 2005; 94: maximum cumulative total of 300 mg kg1 or in the ‘high-dose’ 779–784. group, up to a maximum cumulative total dose of 600 mg kg1. 8 Fujii AM, Patel SM, Allen R, Doros G, Guo CY, Testa S. Poractant alfa and beractant The mean total amount of PA given in the ‘low-dose’ group treatment of very premature infants with respiratory distress syndrome. J Perinatol 2010; 30: 665–670. was 242 mg phospholipid per kg, probably enough to replace 9 Dizdar EA, Sari FN, Aydemir C, Oguz SS, Erdeve O, Uras N et al. A randomized, the entire pulmonary surfactant pool, according to the study controlled trial of poractant alfa versus beractant in the treatment of preterm infants authors. In this study, they did find a significant difference with with respiratory distress syndrome. Am J Perinatol; e-pub ahead of print 21 November 48.4% of babies in the ‘low-dose’ group needing >40% oxygen 2011; PMID 22105435. after 3 days compared with 42.6% of those in the ‘high-dose’ group 10 Fox GF, Sothinathan U. The choice of surfactant for treatment of respiratory distress 29 syndrome in preterm infants: a review of the evidence. Infant 2005; 1: 8–12. (P<0.01). Cogo et al. recently reported on the pharmacokinetics 11 Halliday HL. History of surfactant from 1980. Biol Neonate 2005; 87: 317–322. 1 in pre-term infants treated with 100 or 200 mg kg of PA. The 12 Singh N, Hawley KL, Viswanathan K. Efficacy of porcine versus bovine surfactants for disaturated, phosphatidyl choline (DSPC) half-life was significantly preterm newborns with respiratory distress syndrome: systematic review and meta- longer and fewer babies required additional doses, as well analysis. Pediatrics 2011; 128: e1588–e1595. as improvement in oxygenation index with the higher dose 13 Tonks A, Morris RHK, Price AJ, Thomas AW, Jones KP, Jackson SK. Dipalmitoylpho- of PA. Based on evidence, European Consensus Guidelines sphatidylcholine modulates inflammatory functions of monocytic cells independently of 30 1 mitogen activated protein kinases. Clin Exp Immunol 2002; 124: 86–94. (2010 update) recommended an initial dose of 200 mg kg of 14 Rauprich P, Moller O, Walter G, Herting E, Robertson B. Influence of modified natural PA for early rescue treatment of RDS. Use of higher dose of PA has or synthetic surfactant preparations on growth of bacteria causing infections in the also been endorsed by the European Association of Perinatal neonatal period. Clin Diagn Lab Immunol 2000; 7: 817–822. Medicine. 15 Rudiger M, Tolle A, Meier W, Rustow B. Naturally derived commercial surfactants differ We sincerely thank Dr Cummings for his comments and the in composition of surfactant lipids and in surface viscosity. Am J Physiol Lung Cell Mol Physiol 2005; 288: L379–L383. Journal Editor for giving us the opportunity to clarify all the issues 16 Ru¨diger M, von Baehr A, Haupt R, Wauer RR, Rustow B. Preterm infants that were brought to our attention. with high polyunsaturated fatty acid and plasmalogen content in tracheal aspirates develop bronchopulmonary dysplasia less often. Critical Care Med 2000; 28: 1572–1577. Conflict of interest 17 Poets CF, Arning A, Bernhard W, Acevedo C, von der Hardt H. Active surfactant in The authors reaffirm the COI statement published in our original pharyngeal aspirates of term neonates: lipid biochemistry and surface tension function. article (19). Eur J Clin Invest 1997; 27: 123–128. 18 Bernhard W, Mottaghian J, Gebert A, Rau GA, von der Hardt H, Poets CF. Commercial versus native surfactants; surface activity, molecular components, and the effect of R Ramanathan, JJ Bhatia, K Sekar and FR Ernst calcium. Am J Respir Crit Care Med 2000; 162: 1524–1533. Premier Research Services, Premier Healthcare Alliance, 19 Ramanathan R, Bhatia J, Sekar K, Ernst FR. Mortality in preterm infants with Charlotte, NC, USA respiratory distress syndrome treated with poractant alpha, calfactant, or beractant: a E-mail: [email protected] retrospective study. J Perinatol 2013; 33: 119–125.

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20 Clark RH, Auten RL, Peabody J. A comparison of the outcomes of neonates treated with 26 Gortner L, Pohlandt F, Bartmann P, Bernsau U, Porz F, Hellwege HH et al. High-dose two different natural surfactants. J Pediatr 2001; 139: 828–831. versus low-dose bovine surfactant treatment in very premature infants. Acta Pediatr 21 Holm B, Cummings JJ. Is there evidence for a mortality difference between exogenous 1994; 83: 135–141. surfactant preparations in neonatal RDS? JAP 2008; 2: 78–83. 27 Speer CP, Robertson B, Curstedt T, Halliday HL, Compagnone D, Gefeller O et al. 22 Bhatia J, Saunders WB, Friedlich P, Lavin PT, Sekar KC, York JM et al. Differences Randomized European multicenter trial of surfactant replacement therapy for severe in mortality among infants treated with three different natural surfactants for neonatal respiratory distress syndrome: Single versus multiple doses of Curosurf. respiratory distress syndrome [Abstract]. Pediatr Res 2011 E-PAS2007.617935.16. Pediatrics 1992; 89: 13–20. 23 Trembath AN, Clark RH, Bloom BT, Smith PB, Bose C, Laughon M. Trends in 28 Halliday HL, Tarnow-Mordi WO, Corcoran JD, Patterson CC. Multicentre randomized surfactant use in the United States: changes in clinical practice. e-J Neo Res 2011; 1: trial comparing high and low dose surfactant regimens for the treatment of respiratory 23–30. distress syndrome (the Curosurf 4 trial). Arch Dis Child 1993; 69: 276–280. 24 Muldoon JH. Structure and performance of different DRG classiﬁcation systems for 29 Cogo PE, Facco M, Simanato MG, Rondina C, Baritussio A, Toffolo GM et al. Dosing of neonatal medicine. Pediatrics 1999; 103(1Suppl E): 302–318. porcine surfactant: effect on kinetics and gas exchange in respiratory distress syndrome. 25 Konishi M, Fijiwara T, Naito T, Takeuchi Y, Ogawa Y, Inukai K et al. Surfactant Pediatrics 2009; 124: e950–e957. replacement therapy in neonatal respiratory distress syndrome. A multi-centre, 30 Sweet DG, Carnielli V, Greisen G, Hallman M, Ozek E, Plavka R et al. European randomized clinical trial: comparison of high-versus low-dose of Surfactant-TA. consensus guidelines on the management of neonatal respiratory distress syndrome in Eur J Pediatr 1988; 147: 20–25. preterm infants-2010 Update. Neonatology 2010; 97: 402–417.

In response to mortality in preterm infants with respiratory distress syndrome treated with poractant alfa, calfactant or beractant: a retrospective study

Journal of Perinatology (2013) 33, 165–166; doi:10.1038/jp.2012.163 enrolled. Those techniques cannot correct for systematic differences in patient selection. The paper omits this data, which shows that the populations were different. A large subset of the data presented in the article Mortality in Omission of the length of stay data created an illusory ‘mortality preterm infants with respiratory distress syndrome treated with advantage’ for the surfactant of the study’s sponsor. I wrote Editor poractant alfa, calfactant or beractant: a retrospective study were Lawson in September 2011, identified the omitted data and 1,2 published by the same authors in two abstracts in 2007. The first requested that this paper be retracted as ‘unreliable’ (which is the abstract reported that the Curosurf population has a lower standard for retractions that is used by the Committee on mortality rate than Infasurf or Survanta. The second abstract Publication Ethics, which lists the Journal of Perinatology is a reported that the same Curosurf population had a shorter length of member4). Dr Lawson chose not to retract the paper. The stay than either the Survanta or Infasurf population. From the ‘unreliable’ standard is the proper one for peer-reviewed journals. initial placebo-controlled trials of lung surfactant replacement This paper as e-publishedFwithout the length of stay dataFis at therapy, the mortality improvement due to surfactant therapy least ‘unreliable’ and should be retracted. resulted in an increase, not a decrease, in the length of stay. Preventing death from Respiratory Distress Syndrome will of course produce a much longer length of stay than dying from Respiratory Conflict of interest Distress Syndrome. One report showed the length of stay tripled, I am Chief Medical Officer of ONY, Inc., manufacturer of Infasurf from 20–61 days for surfactant-treated infants compared with (calfactant). placebo.3 How, in this population, was it possible for Curosurf patients to have both a lower mortality and a shorter length of EA Egan stay? The self-evident answer is that Curosurf was administered to Professor of Pediatrics and Physiology and Biophysics, State more patients who had low mortality risk. Similar patients cared University of New York at Buffalo, Buffalo, NY, USA for by physicians who use Infasurf and Survanta are not included E-mail: [email protected] in the database, because they did not receive any surfactant therapy at all. References The article as published, omitted the length of stay data and 1 Bhatia J, Sunders WB, Friedlich PT, Lavin PT, Sekar KC, York JM et al. Differences in reported only the mortality data. It does have multiple regression mortality among infants treated with three different natural surfactants for respiratory analyses ‘correcting’ for possible differences in risk among patients distress syndrome. Pediatric Academic Societies Meeting, E-PAS2007:617935.16, 2007.

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In response to mortality in preterm infants with respiratory distress syndrome treated with poractant alfa, calfactant or beractant: a retrospective study

Journal of Perinatology Letters to the Editor 166

2 Sekar KC, Bhatia J, Ernst FR, Saunders WB, Lavin PT, Ramanathan R et al. 3 Tubman TRJ, Halliday HL, Normand C. Cost of surfactant replacement therapy for severe Resource use in pre term neonates with respiratory distress syndrome (RDS) neonatal respiratory distress syndrome: a randomised controlled trial. Brit Med J 1990; treated with one of three different natural surfactants: analyses using 301: 842–845. a large hospital data base. Acta Pediatr 2007; 96(Suppl 456): p109 4 Retractions: Guidance from the Committee on Publication Ethics. http://publication (Abstract). ethics.org/ﬁles/retraction%20guidelines.pdf.

Response to Dr Egan’s letter

Journal of Perinatology (2013) 33, 166–167; doi:10.1038/jp.2012.171 deathvsBE(P ¼ 0.02) and a significantly shorter LOS (weighted mean difference: 26.3 [95% CI: 36.5 to 16.07]; P<0.00001). In the most recent randomized trial comparing PA with BE, LOS was not Thank you for the opportunity to respond to the letter from Dr Edmund increased in PA group despite a 50% reduction in mortality rate in the A Egan, Chief Medical Officer of ONY, Inc., manufacturer of Infasurf PA-treated group (9.8 vs 20%).8 (Calfactant, (CA)). In the letter by Dr Egan, he refers to the studies In light of the evidence cited above, it is incorrect to assume that presented as abstracts in 2007 (ref 1,2 in his letter) and our study decreased mortality will automatically lead to longer LOS, especially, published electronically as a peer reviewed manuscript in 2011 and when comparing treatment with different surfactants, different timing included in this print issue. These are different studies, in which of administration such as early vs delayed treatment, and different different protocols, different timeframes (abstract 2007: Jan 2003 to June dosessuchashighvslowdose.ItisverylikelythathigherdosesofPA 2006; full publication 2011:Jan 2005 to Dec 2009) different infants, contributed to the positive results seen with PA in all the randomized, different number of patients and different statistical models were used. controlled trials as well as from meta-analysis published to date. Dr Egan discusses the increased length of stay (LOS) resulting from Reasons for shorter LOS accompanying decreased mortality are likely improvement in mortality due to surfactant therapy in studies multifactorial. For example, in six of the randomized trials published comparing surfactant vs placebo. Based on evidence, the introduction to date comparing PA with BE, PA treatment was associated with of surfactant therapy has led to significantly decreased mortality and faster weaning of oxygen and pressures, less need for redosing, earlier morbidity without increasing resource utilization. The statements of extubations, less PDA and air-leaks, and improved survival free of Dr Egan regarding inverse correlation between mortality and LOS are bronchopulmonary dysplasia. All of these secondary outcomes might based on a very old study (ref 3 in his letter) comparing surfactant with havecontributedtotheshorterLOSobserved in PA treated-infants. placebo in a different ‘era’ of neonatology, where respiratory distress Furthermore, we did not omit LOS results to create an illusory syndrome (RDS) was the major cause of death and had a major ‘mortality advantage’ as stated by Dr Egan in his letter. Other influence on LOS. Most important is the fact that surfactant therapy retrospective studies comparing mortality differences between compared with placebo has shown decrease in cost both in infants who animal-derived surfactants do not include LOS amongst covariates survived as well as in infants who died. Subsequent studies on resource in the mortality analysis and therefore our manuscript is entirely utilization are clearly different and these have reported LOS in patients consistent with previous and also subsequent study designs in the who received one of the three animal-derived surfactants.1,2 Baroutis field of retrospective mortality analyses.9,10 In fact, Trembath et al.3 reported decreased mortality and significantly shorter LOS with et al.,10 in a recent report involving 59 342 infants treated with poractant alfa (PA), when compared to alveofact or beractant (BE) in a surfactants concluded that, ‘Poractant alfa and calfactant were randomized, controlled trial. Fujii et al.4 reported a nonsignificant associated with lower incidence of morbidity and mortality as decrease in mortality (8 vs 19%) and shorter LOS in a PA-treated group compared to beractant in premature infants. Given that further compared with that in the BE-treated group (87 vs 97 days, P ¼ 0.179). randomized controlled trials comparing surfactants is unlikely, our In two randomized, controlled trials comparing PA and BE, incidence of findings suggest that the differences in efficacy might be important patent ductus arteriosus (PDA), as well as the need for medical or for clinical practice’. Treatment with PA was associated with fewer surgical ligation of PDA, were significantly less in patients treated with air leaks in the first week vs BE and CA, OR ¼ 0.80 (95% CI; 0.69, PA,4,5 which might therefore contribute to shorter LOS. In a study 0.94) and 0.69 (0.59, 0.82), respectively, and treatment with PA or comparing high vs low dose surfactant therapy, LOS was shorter in the CA vs BE was associated with lower incidence of BPD, OR ¼ 0.92 high dose group when compared with low dose group (82 vs 99 days).6 (0.88, 0.95) and 0.89 (0.85, 0.93) and mortality, OR ¼ 0.71 (0.65, A recently published systematic review and meta-analysis,7 demonstrated 0.77) and 0.92 (0.85, 0.99), respectively, which persisted after a good correlation between higher surfactant efficacy (mortality adjustment.10 These study findings are entirely consistent with our reduction) and reduced LOS with poractant alfa (PA). Indeed, in the study findings and other randomized studies involving PA and BE. meta-analysis, PA was associated with both a significant reduced risk of Furthermore, we have clearly stated in our previous response letter

Journal of Perinatology Letters to the Editor 166

Response to Dr Egan’s letter

Journal of Perinatology Letters to the Editor 167 to Dr Cummings, the reasons for not including LOS results in our 2 Schwartz RM, Luby AM, Scanlon JW, Kellogg RJ. Effect of surfactant on morbidity, manuscript discussing mortality as the primary outcome for this mortality, and resource use in newborn infants weighing 500 to 1500 g. N Engl J Med study. We used appropriate statistical methods that are typically 1994; 330: 1476–1480. used with real-world outcomes studies involving large databases. 3 Baroutis G, Kaleyias J, Liarou T, Papathoma E, Hatzistamatiou Z, Costalos C. Comparison of three treatment regimens of natural surfactant preparations in neonatal We conducted this study in an ethical manner without any respiratory distress syndrome. Eur J Pediatr 2003; 162: 476–480. influence whatsoever from the study sponsor and it was subjected 4 Fujii AM, Patel SM, Allen R, Doros G, Guo C-Y, Testa S. Poratcant alfa and beractant to a rigorous peer review process, similar to any other manuscript. treatment of very premature infants with respiratory distress syndrome. J Perinatol 2010; 1–6. 5 Malloy CA, Nicoski P, Muraskas JK. A randomized trial comparing beractant and Conflict of interest poractant treatment in neonatal respiratory distress syndrome. Acta Paediatr 2005; 94(6): 779–784. The authors declare no conflict of interest. 6 Gortner L, Pohlandt F, Bartmann P, Bernsau U, Porz F, Hellwege HH et al. High dose versus low dose bovine surfactant treatment in very premature infants. Acta Pediatr R Ramanathan, JJ Bhatia, K Sekar and FR Ernst 1994; 83: 135–141. Premier Research Services, Premier Healthcare Alliance, 7 Singh N, Hawley KL, Viswanathan K. Efficacy of porcine versus bovine surfactants Charlotte, NC, USA for preterm newborns with respiratory distress syndrome: Systematic review and E-mail: [email protected] meta-analysis. Pediatrics 2011; 128: e1588–e1595. 8 Dizdar EA, Sari FN, Aydemir C, Oguz SS, Erdeve O, Uras N et al. A randomized, controlled trial of poractant alfa versus beractant in the treatment of preterm infants with respiratory distress syndrome. Am J Perinatol 2012; 29(2): 95–100. References 9 Clark RH, Auten RL, Peabody J. A comparison of the outcomes of neonates treated with 1 Sekar KC, Bhatia J, Ernst FR et al. Resource use in preterm neonates with respiratory two different natural surfactants. J Perinatol 2001; 139: 828–831. distress syndrome (RDS) treated with one of the three different natural surfactants: 10 Trembath A, Hornik C, Clark R, Smith P, Laughon M, Beitzel M. Comparative analyses using a large hospital data base. Acta Pediatr 2007; 96(Suppl 456): p109 effectiveness of three surfactant preparations in premature infants. Pediatr Res 2012; (Abstract). 1535: 617A.

Journal of Perinatology Journal of Perinatology (2013) 33, 126–133 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Efﬁcacy of phototherapy devices and outcomes among extremely low birth weight infants: multi-center observational study

BH Morris1, JE Tyson2, DK Stevenson3,WOh4, DL Phelps5, TM O’Shea6, GE McDavid2, KP Van Meurs3, BR Vohr4, C Grisby7, Q Yao8, S Kandefer8, D Wallace8 and RD Higgins9 1Department of Neonatology, Trinity Mother Frances Health System, Tyler, TX, USA; 2Department of Pediatrics, University of Texas Medical School at Houston, Houston, TX, USA; 3Stanford University School of Medicine, Palo Alto, CA, USA; 4Department of Pediatrics, Women and Infants Hospital, Brown University, Providence, RI, USA; 5University of Rochester School of Medicine and Dentistry, Rochester, NY, USA; 6Wake Forest University School of Medicine, Winston-Salem, NC, USA; 7Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA; 8Statistics and Epidemiology Unit, RTI International, Research Triangle Park, NC, USA and 9Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

Introduction Objective: Evaluate the efficacy of phototherapy (PT) devices and the The first phototherapy (PT) device used fluorescent bulbs and was outcomes of extremely premature infants treated with those devices. described in 1958.1 PT has long since become the primary therapy Study Design: This substudy of the National Institute of Child Health for hyperbilirubinemia in neonates. Current options for PT include and Human Development Neonatal Research Network PT trial included conventional fluorescent lights (Banks), halogen spotlights (Spots), 1404 infants treated with a single type of PT device during the first fiberoptic blankets (Blankets) and the relatively new blue 24±12 h of treatment. The absolute (primary outcome) and relative light-emitting diode (LED) lights.2 The theoretical advantages decrease in total serum bilirubin (TSB) and other measures were of LED lights include a narrow light spectrum in the blue range, evaluated. For infants treated with one PT type during the 2-week minimal heat production, power efficiency and low-maintenance intervention period (n ¼ 1223), adjusted outcomes at discharge and 18 requirements.3 to 22 months corrected age were determined. Although these types of PT devices are used routinely in Result: In the first 24 h, the adjusted absolute (mean (±s.d.)) and neonatal units around the world, there are relatively few studies relative (%) decrease in TSB (mg dl1) were: light-emitting diodes in premature infants. A few small studies have compared the (LEDs) 2.2 (±3), 22%; Spotlights 1.7 (±2), 19%; Banks 1.3 effectiveness of conventional PT with Blankets in premature 4–9 (±3), 8%; Blankets 0.8 (±3), 1%; (P<0.0002). Some findings at infants. Comparisons of LEDs with other types of PT have been 10–13 18 to 22 months differed between groups. limited to small trials of term or preterm infants. For extremely low birth weight (ELBW) infants, very little is known Conclusion: LEDs achieved the greatest initial absolute reduction in TSB about the efficacy of the various PT devices under ‘real world’ but were similar to Spots in the other performance measures. Long-term conditions or any effects on clinical outcomes. Important effects on effects of PT devices in extremely premature infants deserve rigorous later outcomes would be most likely seen in ELBW infants whose evaluation. skin is relatively translucent and would allow deeper penetration of Journal of Perinatology (2013) 33, 126–133; doi:10.1038/jp.2012.39; the PT lights.14 published online 12 April 2012 The National Institute of Child Health and Human Development Keywords: extremely low birth weight; neonatal jaundice; neurodevel- (NICHD) Neonatal Research Network conducted a randomized trial opmental outcome; phototherapy comparing the use of aggressive PT with more conservative PT in ELBW infants.15 We used data from this trial to perform a prospective non-randomized comparison of the effectiveness of different PT devices in the ELBW population. Our primary outcome was the absolute decrease in total serum bilirubin (TSB) in the first Correspondence: Dr BH Morris, Department of Neonatology, Trinity Mother Frances Health 24 h of treatment. Secondary outcomes included length of PT, System, 800 East Dawson Street, Tyler, TX 75703, USA. irradiance levels, proportion of infants whose TSB increased to E-mail: [email protected] 1 Received 2 December 2011; revised 24 February 2012; accepted 24 February 2012; published within 2 mg dl of the exchange transfusion criterion, the online 12 April 2012 incidence of medical morbidities and adverse neurodevelopmental Phototherapy devices and ELBW infants BH Morris et al 127 outcomes. Based on their theoretical advantages, we hypothesized and refusal of consent by parents. Therefore, the TSB within 4 h that LEDs would produce the largest drop in TSB during the first before the initial start of PT was collected and then repeated within 24 h of treatment. No specific hypotheses were formulated about 24±12 h in 1142 of the infants. The mean absolute and relative other outcomes. change in TSB was calculated for each device group. We considered a decrease in TSB of >1.5 mg dl1 to be clinically significant and calculated the proportion of infants attaining this in the first 24 h Methods for each device group. The Neonatal Research Network trial was approved by the The criteria for starting PT, for performing an exchange Institutional Review Board of all the participating centers (see transfusion and for stopping and restarting PT during the first 14 Appendix). Written informed consent was obtained for all infants days were previously described.15 The medical staff selected the type (n ¼ 1974). Infants were stratified by birth weight (501 to 750 g of PT device. Any time PT was started it was continued for a and 751 to 1000 g) and center and then randomized to either minimum of 24 h. PT was intensified at TSB values of 11 mg dl1 aggressive or conservative PT. For this substudy, we excluded for infants 501 to 750 g and 13 mg dl1 for infants 751 to 1000 g. infants who were initially treated with more than one type of The definition of ‘high TSB’ was predefined as a TSB within PT light (n ¼ 90), had type of PT-light information missing 2mgdl1 of the predetermined exchange transfusion criterion (n ¼ 264) or received no PT in the main trial (n ¼ 216). (X11 mg dl1 for 501 to 750 g infants and X13 mg dl1 for 751 The remaining 1404 infants were included in evaluating the to 1000 g infants). By protocol, an exchange transfusion was performance of the PT devices in the first 24±12 h. (Figure 1) indicated in both the treatment groups if the TSB exceeded the Medical morbidities and neurodevelopmental outcomes were threshold values after 8 h of intensified treatment. Among all the evaluated in the 1223 infants who were treated with only one type infants in the main trial, two infants in the aggressive cohort and of PT during the 2-week intervention period. three infants in the conservative cohort received an exchange The absolute decrease in TSB in the first 24 h was selected as transfusion. the primary outcome because it was considered the most direct Devices were categorized as Banks, Blankets, Spots and measure of the efficacy of the PT device. The pretreatment TSB was LEDs. The irradiance levels were maintained between 15 and mandatory in the first 200 infants enrolled in the PT trial but was 40 mWcm2 nm and were monitored by research nurses on subsequently optional because of concerns of excessive blood loss weekdays and by bedside nurses per nursery routine. Irradiance was

1974 Infants enrolled in main trial

90- Infants treated with > 1 type of light 264- Infants with insufficient records 216- Infants did not receive PT

1404 eligible for this study

LED Blankets Banks Spots Initial PT 364 Initial PT 141 Initial PT 435 Initial PT 464 Exclusive 307 Exclusive 117 Exclusive 378 Exclusive 421

Died 64 Survived 243 Died 38 Survived 79 Died 86 Survived 292 Died 78 Survived 343

Figure 1 Enrollment, PT type and survival of study patients.

Journal of Perinatology Phototherapy devices and ELBW infants BH Morris et al 128 measured at the umbilicus for supine infants and the lumbar area (continuous)), and the dichotomous measure of 5 min Apgar score for prone infants using an Ohmeda Biliblanket Meter. (Biliblanket <5. Each model was initially fit with a device by treatment arm meter no. 66000198-900; Ohmeda Medical, Laurel, MD, USA). To interaction to assess the potential effect modification of treatment minimize handling of infants treated with Blankets, the irradiance on device type. Because these models showed no evidence of an was measured only on the first day. The mean irradiance level was interaction, only the results for the main effect of device-type determined for each device group among the 1223 infants who controlling for treatment are presented. For binary outcome received only one type of PT light throughout the intervention measures, an overall test of device effect and adjusted relative period. risk (RR) estimates (with LEDs as the reference category) were Other secondary outcomes were determined for infants treated calculated using robust Poisson regression in a generalized with only one type of PT for the duration of the 14-day study estimating equation model.17 For continuous outcome measures, period. The secondary outcomes evaluated and included the total the overall test of device effect and adjusted device-specific means duration of PT, the proportion of infants who reached a high TSB, were obtained using general linear models. No adjustments were medical morbidities and neurodevelopmental outcomes as made for multiple comparisons. All the analyses were conducted determined by trained and certified examiners. Pre-discharge using SAS (v 9.2) software (SAS Institute, Cary, NC, USA). medical morbidities recorded by research nurses using pre-specified definitions included bronchopulmonary dysplasia (oxygen administration at 36 weeks post menstrual age), patent ductus arteriosus (PDA), retinopathy of prematurity, intraventricular Results hemorrhage grade 3 or 4, necrotizing enterocolitis and death.15 The number and proportion of infants initially treated with each Neurodevelopmental impairment at 18 to 22 months corrected age PT device were: Spots 464 (33%), Banks 435 (31%), LEDs 364 was defined as at least one of the following: blindness (no (26%) and Blankets 141 (10%). The baseline demographics and functional vision in either eye), severe hearing loss (any hearing perinatal variables are listed in Table 1. Race was significantly loss with bilateral hearing aids prescribed), moderate or severe different between device types (P<0.001). Marginally significant cerebral palsy, or Bayley Scales of Infant Development II mental differences were observed for birth weight and gestational age. development index (MDI) or psychomotor development index <70 Although the adjusted model showed statistically significant (two s.d. below the mean).16 differences in the pretreatment TSB across PT device types, the small differences have doubtful clinical importance (Table 2). Statistical analysis The differences in the mean TSB levels 24±12 h after initial The demographic and perinatal variables were compared by using start of PT, the absolute decrease (primary outcome) and relative analysis of variance for continuous variables and w2 tests for decrease in TSB across the PT devices were statistically significant categorical variables. Model-based analyses were used to obtain in unadjusted and adjusted models. The adjusted absolute decrease adjusted estimates of device effects after controlling for treatment in TSB for LEDs (2.2 mg dl1) was significantly greater than group, stratifying variables (center and birth weight group) and that for Spots (1.7 mg dl1; P ¼ 0.0038), Banks (1.3 mg dl1; other potential confounders (sex, race and gestational age P<0.0001) and Blankets (0.8 mg dl1; P<0.001). The adjusted

Table 1 Baseline characteristics

LEDs n ¼ 364 Blankets n ¼ 141 Banks n ¼ 435 Spots n ¼ 464 P-value

Birth weight (g)a 771±136 784±138 766±137 789±128 0.05 Gestational age (weeks)a 25.8±1.9 26.1±1.9 25.8±1.9 26.1±1.9 0.07

Race n (%) <0.001 Black 156 (43) 90 (64) 188 (43) 142 (31) White 109 (30) 44 (31) 121 (28) 239 (52) Hispanic/other 99 (27) 7 (5) 126 (29) 83 (18)

Male n (%) 192 (53) 63 (45) 222 (51) 241 (52) 0.42 5 min Apgar <5 n (%) 58 (16) 13 (9) 52 (12) 63 (14) 0.17 Age at start of PT (h)a 35±22 34±25 36±25 36±23 0.62

Abbreviations: Banks, ﬂuorescent lights; Blankets, ﬁberoptic blankets; LED, light-emitting diode; Spots, halogen spotlights. aMean±s.d.

Journal of Perinatology Phototherapy devices and ELBW infants BH Morris et al 129 a absolute decrease in TSB for Spots was signiﬁcantly greater than -value P that for Blankets (P ¼ 0.007) and Banks (P ¼ 0.042). The adjusted absolute decrease in TSB for Banks compared with that for Blankets was similar. LEDs and Spots had a similar adjusted 3 0.04 2 <0.0001 2 <0.0001

9 <0.0001 relative decrease in TSB (22% and 19%, respectively) and both 44 <0.001 ± ± ± 19% <0.01 ± ±

1.7 were signiﬁcantly greater than Banks (8%; vs LEDs P ¼ 0.0025; vs Spots P ¼ 0.032) and Blankets (1%; vs LEDs P ¼ 0.0087; vs 3 7.3 2 5.6 3 625 45 70 ± ± ± Spots P 0.015).

8% ¼ ± ± 1.3 The unadjusted and adjusted proportion of infants in each 1 3 7.5 2 6.2 3 group with >1.5 mg dl decrease in TSB in the ﬁrst 24 h of 818 55 76 ± ± ± 1% ±

± treatment differed among the groups. LEDs were more likely to 0.8

achieve this decrease than either Blankets or Banks (P ¼ 0.0006 3 6.9 2 6.3 3 626 40 85 and P 0.0066, respectively) and Spots were more likely to achieve ± ± ± ¼ 22% ± ± 2.2 this decrease than Blankets (P ¼ 0.0070). The unadjusted proportions of infants who reached a high TSB were similar

406) 65 between the device groups (P ¼ 0.74). The adjusted analyses could ¼ 464) 23 3 7.5 2 2 5.2 n not be performed for this variable because of the small number of ¼ ± ± ± n 13%

1.5 events in each group. 9( ominator for each outcome was the number of infants for whom the result was known.

± The mean irradiance levels over the intervention period were 44; (58) (

± statistically different with Banks having the lowest irradiance of all

alogen spotlights; TSB, total serum bilirubin. groups. Spots and Blankets were statistically similar and had the

358) 69 highest mean irradiance levels. (Table 2) The adjusted mean ¼ 435) 24 3 7.0 3 2 5.4 n duration of PT treatment was different between the device groups. ¼ ± ± ± 3% n LEDs had the shortest duration of PT, signiﬁcantly less than 1.1 6(

± Blankets or Banks (P ¼ 0.0011 and P ¼ 0.0014, respectively), but 42; (57) (

± not different from Spots (P ¼ 0.18). The adjusted duration of PT for Blankets and Banks was statistically similar.

102) 68 Over the 14-day intervention period, 1223 infants received a ¼ 141) 20 3 6.7 3 2 5.6 n single type of PT: LEDs 307 (25%), Blankets 117 (10%), Banks 378 ¼ ± ± ± 4% n (31%) and Spots 421 (34%). The medical outcomes for these 0.4 9( infants prior to discharge are shown in Table 3. After adjusting for ± 56; (57) (

± the variables in our model, the only statistically signiﬁcant comparisons were a decreased RR for PDA (RR (95% conﬁdence

301) 85 interval (CI)) 0.65 (0.44, 0.96)) and PDA or death (RR (95% CI) ¼ 364) 27 3 6.8 3 2 6.5 n 0.68 (0.49, 0.92)) for infants treated with Blankets compared ¼ ± ± ± n 52% 28% 38% 44% 58% 23% 40% 47% <0.01 18% with LEDs. In adjusted results not shown in Table 3, Blankets 2.1 LEDs Blankets Banks Spots LEDs Blankets Banks Spots 16 (4) 13 (9) 18 (4) 16 (4) NA NA NA NA NA 6( also showed a decreased RR for PDA compared with Banks ± 40; (54) ( 24

± (RR (95% CI) 0.55 (0.37, 0.82)) and Spots (RR (95% CI) 0.66 (0.46, 0.95)) and a decreased RR for PDA or death compared with d Banks (RR (95% CI) 0.60 (0.44, 0.83)) and Spots (RR (95% CI) 1142) 1142) ¼

1142) 0.69 (0.51, 0.92)). For necrotizing enterocolitis, there was a 1325) 4.9 ¼ d n n ¼ 1330) 66 ¼ ( decreased RR for Blankets compared with Spots (RR (95% CI) 0.40 n n ¼ ( b,c 1392) n (0.17, 0.96)). b,c ¼ 12 h 12 h ( n The infant outcomes at 18 to 22 months corrected age are ± ± drop in TSB ( 1198) 7.2 (%) (

1 shown in Table 4. For death or neurodevelopmental impairment . 1 1392) ¼ n n (Median–h) ( (primary outcome of main trial), neither the unadjusted nor the ¼ ( b n ( b,c adjusted analyses showed a difference in risk among the PT b

12 h after starting PT devices. The only significant infant outcomes were in relation to nm) Total serum bilirubin and phototherapy results 2 ± s.d. MDI <85 and death or MDI <85. For MDI <85, Blankets were ± associated with a decreased risk compared with LEDs (RR (95% CI) (proportion). Wcm m Model adjusted for center,Mean birth weight group, treatment group, device type, sex,n race, 5 min APGAR <5 (yes/no) and gestational age (continuous). Den TSB measured in mg dl ( Duration of PT (h) NA, adjusted model could not be performed for this variable because of small number of events. Table 2 VariableAbbreviations: Banks, fluorescent lights; Blankets, fiberoptica blankets; LED, light-emitting diode;b NA, not available;c PT, phototherapy; Spots, h d Unadjusted means or proportions Adjusted means or proportions Adjusted Mean irradiance levels in first 14 days Infants with high TSB Pretreatment TSB TSB at 24 Absolute change in TSB at 24 Relative change in TSB at 24 Infants with >1.5 mg dl 0.68 (0.48, 0.96)), whereas Banks showed an increased risk

Journal of Perinatology Phototherapy devices and ELBW infants BH Morris et al 130

Table 3 Outcomes at discharge for infants treated with a single type of phototherapy

Outcome Phototherapy device n (%) Adjusted RR (95% CI)a

LEDs Blankets Banks Spots Blankets/LEDs Banks/LEDs Spots/LEDs

Death within 14 days of birth 29 (9) 16 (14) 39 (10) 20 (5) 0.80 (0.31, 2.06) 1.10 (0.62, 1.97) 0.60 (0.31, 1.17) Death before discharge 63 (21) 36 (31) 75 (20) 69 (16) 0.84 (0.45, 1.55) 0.77 (0.53, 1.12) 0.89 (0.60, 1.32) IVH grade 3 or 4 80 (26) 28 (25) 95 (25) 65 (16) 0.81 (0.46, 1.42) 0.92 (0.66, 1.28) 0.70 (0.49, 1.02) Death or IVH grade 3 or 4 99 (32) 42 (36) 118 (31) 86 (21) 0.89 (0.55, 1.44) 0.95 (0.71, 1.26) 0.73 (0.53, 1.01) BPD at 36 weeks 112 (45) 44 (51) 145 (46) 150 (42) 0.81 (0.55, 1.20) 1.00 (0.79, 1.26) 0.89 (0.69, 1.14) Death or BPD at 36 weeks 167 (55) 73 (63) 205 (54) 209 (51) 0.94 (0.71, 1.24) 1.00 (0.84, 1.18) 0.95 (0.79, 1.14) PDA 155 (50) 39 (33) 216 (57) 195 (46) 0.65 (0.44, 0.96)* 1.17 (0.98, 1.41) 0.99 (0.80, 1.22) Death or PDA 179 (58) 58 (50) 245 (65) 222 (53) 0.68 (0.49, 0.92)* 1.12 (0.96, 1.31) 0.99 (0.82, 1.18) NEC 36 (12) 8 (7) 36 (10) 65 (15) 0.44 (0.17, 1.11) 1.18 (0.69, 2.02) 1.10 (0.67, 1.82) Death or NEC 83 (27) 39 (33) 94 (25) 108 (26) 0.69 (0.41, 1.17) 0.95 (0.70, 1.31) 1.03 (0.74, 1.43) ROP stage 3–5 48 (19) 20 (24) 65 (21) 67 (19) 1.73 (0.94, 3.18) 1.50 (0.99, 2.27) 1.13 (0.73, 1.75) Death or ROP stage 3–5 110 (36) 55 (50) 136 (37) 132 (33) 1.23 (0.84, 1.82) 1.11 (0.87, 1.42) 0.98 (0.76, 1.27)

Abbreviations: Banks, fluorescent lights; Blankets, fiberoptic blankets; BPD, bronchopulmonary dysplasia; CI, confidence interval; IVH, intraventricular hemorrhage; LED, light-emitting diode; NEC, necrotizing enterocolitis; PDA, patent ductus arteriosus; ROP, retinopathy of prematurity; RR, relative risk; Spots, halogen spotlights; TSB, total serum bilirubin. aModel adjusted for center, birth weight group, treatment group, device type, sex, race, 5 min APGAR <5 (yes/no) and gestational age (continuous). Device type by treatment interaction was not significant and hence not included in the model. *P<0.05, denominator for each outcome was the number of infants for whom the result was known.

Table 4 Outcomes at 18–22 months for infants treated with a single type of phototherapy

Outcome Phototherapy device n (%) Adjusted RR (95% CI)a

LEDs Blankets Banks Spots Blankets/LEDs Banks/LEDs Spots/LEDs

Death or NDI 87 (39) 24 (35) 96 (37) 102 (35) 0.96 (0.69, 1.33) 0.97 (0.81, 1.18) 1.01 (0.82, 1.25) NDI 151 (52) 62 (58) 182 (53) 180 (49) 0.89 (0.53, 1.50) 0.94 (0.70, 1.27) 0.92 (0.65, 1.31) Death 64 (21) 38 (33) 86 (24) 78 (20) 0.86 (0.47, 1.55) 0.95 (0.67, 1.35) 1.05 (0.72, 1.54) Moderate/severe CP 11 (5) 3 (4) 16 (6) 26 (9) 0.81 (0.25, 2.68) 0.98(0.44, 2.17) 1.18 (0.56, 2.49) Death or moderate/severe CP 75 (25) 41 (37) 102 (28) 104 (27) 0.90 (0.54, 1.50) 0.97 (0.71, 1.32) 1.11 (0.80, 1.54) Severe hearing lossb 8 (4) 1 (1) 4 (1) 10 (3) 0.47 (0.07, 3.31) 0.49 (0.16, 1.51) 0.92 (0.38, 2.25) Death or severe hearing loss 72 (25) 39 (35) 90 (25) 88 (23) 0.72 (0.42, 1.24) 0.87 (0.62, 1.22) 0.92 (0.64, 1.32) MDI <70 70 (31) 19 (28) 77 (30) 76 (26) 0.76 (0.40, 1.43) 0.96 (0.68, 1.37) 0.85 (0.55, 1.31) Death or MDI <70 134 (46) 57 (53) 163 (47) 154 (41) 0.90 (0.63, 1.31) 0.99 (0.80, 1.22) 1.01 (0.79, 1.28) MDI <85 139 (62) 36 (52) 160 (62) 153 (52) 0.68 (0.48, 0.96)* 1.23 (1.02, 1.49)* 0.80 (0.64, 1.00) Death or MDI <85 203 (70) 74 (69) 246 (71) 231 (62) 0.81 (0.64, 1.02) 1.16 (1.02, 1.33)* 0.92 (0.79, 1.07) PDI <70 47 (21) 15 (22) 52 (20) 70 (24) 1.38 (0.68, 2.81) 1.06 (0.67, 1.67) 1.36 (0.83, 2.23) Death or PDI <70 111 (39) 53 (50) 138 (40) 148 (40) 1.13 (0.76, 1.69) 1.02 (0.80, 1.30) 1.17 (0.90, 1.52) PDI <85 95 (43) 58 (78) 100 (38) 126 (43) 0.95 (0.59, 1.54) 1.14 (0.86, 1.51) 1.10 (0.80, 1.51) Death or PDI <85 159 (56) 64 (60) 186 (54) 204 (55) 1.00 (0.74, 1.36) 1.08 (0.90, 1.29) 1.11 (0.91, 1.35) Normal gross motor function 177 (77) 58 (78) 213 (78) 232 (77) 1.13 (0.92, 1.39) 1.06 (0.95, 1.20) 1.05 (0.92, 1.20) Walk ﬂuently 177 (77) 56 (76) 204 (75) 226 (75) 0.98 (0.79, 1.21) 0.97 (0.86, 1.10) 0.94 (0.82, 1.08) Fine pincer grasp 197 (85) 63 (85) 231 (85) 254 (84) 1.08 (0.92, 1.28) 1.09 (0.99, 1.19) 1.05 (0.94, 1.16)

Abbreviations: Banks, fluorescent lights; Blankets, fiberoptic blankets; CI, confidence interval; CP, cerebral palsy; LED, light-emitting diode; MDI, mental development index; NA, not available; NDI, neurodevelopmental impairment; PDI, psychomotor development index; RR, relative risk; Spots, halogen spotlights; TSB, total serum bilirubin. aModel adjusted for center, birth weight group, treatment group, device type, sex, race, 5 min APGAR <5 (yes/no) and gestational age (continuous). Device type by treatment interaction was not significant and hence not included in the model. bBecause of rarity of outcome, center was excluded from the model. *P<0.05, denominator for each outcome was the number of infants for whom the result was known. compared with LEDs (RR (95% CI) 1.23 (1.02, 1.49)). In results (1.21, 1.96)). For death or MDI <85, Banks showed an increased not shown in Table 4, Blankets had a decreased risk of MDI <85 risk compared with the other devices: LEDs (RR (95% CI) 1.16 compared with Banks (RR (95% CI) 0.55 (0.39, 0.79)) and Banks (1.02, 1.33)), Spots (RR (95% CI) 1.26 (1.08, 1.48)) and Blankets had an increased risk compared with Spots (RR (95% CI) 1.54 (RR (95% CI) 1.43 (1.12, 1.85)).

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Discussion comparisons were performed. It is reassuring to know that for Among PT devices used to treat hyperbilirubinemia in this large death or neurodevelopmental impairment (primary outcome of the group of ELBW infants, LEDs were associated with the largest NICHD PT trial) and almost all of the other outcomes, there were absolute decrease in TSB in the first 24±12 h of PT. LEDs and no significant differences in risk among the PT devices. However, Spots were similar for the relative decrease in TSB and duration of the wide CIs for this and other outcomes do not exclude the treatment. LEDs performed better than Banks and Blankets but possibility of important differences in the long-term effects of these similar to Spots in reducing TSB >1.5 mg dl1 within 24±12 h of commonly used devices. PT. Overall, LEDs and Spots appeared to have similar effectiveness There were a few differences that deserve some discussion. and duration of treatment. This information could be important Infants treated with Blankets were at lower risk for PDA and PDA or for clinicians starting PT on ELBW infants with a high TSB where death compared with those treated with the other PT devices. There a rapid response is desired to minimize the possibility of bilirubin was also a decreased risk of NEC with Blankets compared with encephalopathy, need for an exchange transfusion and exposure Spots, however, this was not found for the combined outcome of NEC or death. Could there be different physiological effects for an to PT. The LED devices were effective despite a relatively lower ELBW infant lying on their back on a biliblanket vs an overhead irradiance probably because the emitted light is in the maximally PT light? There have been trials showing a hemodynamic effect effective range of 450 to 470 nm and not diluted with other from PT. In a small randomized trial, chest shielding during PT wavelengths that would be included in the measurement range reduced the frequency of PDA and length of hospital stay,22 but in (400 to 520 nm) of the Ohmeda Biliblanket Meter. another trial this effect was not found.23 Benders et al24,25 has Maintaining and monitoring irradiance levels will be important shown hemodynamic changes in term and premature infants while for obtaining similar results in clinical practice. The NICHD PT on PT, which resolved when PT was stopped. Pezzati et al26and Yao trial had a PT target range of 15 to 40 mWcm2 nm which et al27 have shown changes to the postprandial mesenteric blood was significantly higher than the estimated irradiance of 6 to flow with conventional PT. Pezzatti et al did not find these blood 10 mWcm2 nm used in the first NICHD PT trial.18 This irradiance flow changes with fiber-optic PT. The recent NICHD PT trial did may be higher than some older PT devices can attain. Even with not show a difference in the RR of PDA or death (RR (95% CI) the newer PT lights, the positioning of the light and distance from 0.95, (0.88, 1.02)), PDA (RR (95% CI) 0.93 (0.86, 1.02)) or NEC the infant can dramatically affect the achieved irradiance levels 19 (RR (95% CI) 0.9 (0.70, 1.14)) between the aggressive and and the surface area affected. conservative groups despite a very significant difference in the In this study, the poor performance of the Banks could be duration of PT treatment (88 vs 35 h, P<0.001).15 These results in related to the relatively low irradiance levels achieved either from a large randomized trial would argue against a causal association older PT units or positioning. The Blankets had the highest mean between PT and PDA or NEC. irradiance level but were not very effective. A potential explanation An important outcome associated with bilirubin encephalopathy for this could be the skin surface area irradiated by the Blankets. 20 is hearing loss. We could detect no difference between the device Dicken estimated that the fraction of the total surface area groups in the prevalence of severe hearing loss or severe hearing irradiated by a Blanket to be 6% for a term infant and 9% for a loss and death. However, hearing loss was an infrequent outcome preterm infant compared with 33% for overhead banks. Tayman limiting our power to detect associations. recently compared overhead and underneath LED devices with The only outcomes at 18 to 22 months corrected age that 2 21 identical irradiances (30 mWcm nm) in infants X35 weeks. differed among the PT device groups were MDI <85 and death or The results demonstrated that the overhead units were more MDI <85. The results suggest that infants treated with Banks had effective in reducing TSB levels and had a shorter duration of PT. an increased risk for either of these outcomes compared with They theorized that the overhead unit illuminated a larger surface the other three device types. In addition, Blankets had a decreased area resulting in better efficacy. Our findings would support the risk for MDI <85 compared with LEDs but not for the composite concept of an overhead PT system being more effective than a outcome of death or MDI <85. We do not have a plausible system below the infant given similar irradiance levels. biological theory to explain these findings. These differences In evaluating therapies, it is important to consider clinical and were not hypothesized before the study and due to the large neurodevelopmental outcomes beyond discharge especially in this number of comparisons performed could be because of chance. vulnerable population. The NICHD Neonatal Research Network PT PT is administered to the great majority of ELBW infants. This trial provided an opportunity to explore the possibility that different study represents the largest sample of ELBW infants treated with PT PT lights may have divergent effects. However, our findings here and analyzed to assess the performance of the devices and the should be interpreted with caution because the assignment of PT outcomes of the infants. LED lights were the most efficient at devices was not randomized, which introduced a potential for bias reducing the absolute TSB in the first 24±12 h of treatment but and created device groups of different sizes, and a large number of were similar to Spots in the other performance measures. Our

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findings indicate short-term differences in the efficacy of the 9 Van Kaam AHLC, van Beek RHT, Vergunst-van Keulen JG, van der Heijden J, Lutz- devices used to administer this therapy and the possibility of Dettinger N, Hop W et al. Fiber optic versus conventional phototherapy for important differences at 18 to 22 months. Different PT devices hyperbilirubinemia in preterm infants. Eur J Pediatr 1998; 157: 132–137. being developed or used in treating high-risk infants deserve 10 Bertini G, Perugi S, Elia S, Pratesi S, Dani C, Rubaltelli FF. Transepidermal water loss and cerebral hemodynamics in preterm infants: conventional versus LED phototherapy. rigorous testing in randomized trials that include both short- and Eur J Pediatr 2008; 167: 37–42. long-term follow-up assessments. 11 Martins BMR, de Carvalho M, Moreira ME, Lopes JMA. Efficacy of new microprocessed phototherapy system with five high density light emitting diodes (Super LED). J Pediatr (Rio J) 2007; 83: 253–258. Conflict of interest 12 Seidman DS, Moise J, Ergaz Z, Laor A, Vreman HJ, Stevenson DK et al. A new blue light-emitting phototherapy device: a prospective randomized controlled study. Natus Medical loaned light-emitting diode phototherapy lights J Pediatr 2000; 136(6): 771–774. to each center. These lights were used at the discretion of the 13 Maisels MJ, Kring EA, DeRidder J. Randomized controlled trial of light-emitting diode attending neonatologist in treating infants in either treatment phototherapy. J Perinatol 2007; 27: 565–567. group. The lights were returned to Natus Medical or purchased at a 14 Kaplan M, Gold V, Hammerman C, Hochman A, Goldschmidt D, Vreman HJ et al. prorated price after the study. Natus Medical has no role in the Phototherapy and photo-oxidation in premature neonates. Biol Neonate 2005; 87: study design, data collection, data analysis or manuscript 44–50. 15 Morris BH, Oh W, Tyson JE, Stevenson DK, Phelps DL, O’Shea TM et al. Aggressive vs preparation or revision. conservative phototherapy for infants with extremely low birth weight. N Engl J Med 2008; 359: 1885–1896. 16 Bayley N. Bayley Scales of Infant Development II. The Psychological Corporation: San Acknowledgments Antonio, TX, 1993. 17 Spiegelman D, Hertzmark E. Easy SAS calculations for risk or prevalence This work was supported by grants from the National Institutes of Health and ratios and differences. Invited Editorial Note. Am J Epidemiol 2005; 162(3): the Eunice Kennedy Shriver National Institute of Child Health and Human 199–200. Development, which provided oversight for study conduct. 18 Landry RJ, Scheidt PC, Hammond RW. Ambient light and phototherapy conditions of eight neonatal care units: a summary report. Pediatrics 1985; 75: 434–436. References 19 Hart G, Cameron R. The importance of irradiance and area in neonatal phototherapy. Arch Dis Child Fetal Neonatal Ed 2005; 90: F437–F440. 1 Cremer RJ, Perryman PW, Richards DH. Influence of light on the hyperbilirubinemia 20 Dicken P, Grant LJ, Jones S. An evaluation of the characteristics and performance of of infants. Lancet 1958; 271: 1094–1097. neonatal phototherapy equipment. Physiol Meas 2000; 21: 493–503. 2 Vreman HJ, Wong RJ, Stevenson DK, Route RK, Reader SD, Fejer MM et al. Light- 21 Tayman C, Tatli MM, Aydemir S, Karadag A. Overhead is superior to underneath light- emitting diodes: a novel light source for phototherapy. Pediatr Res 1998; 44(5): emitting diode phototherapy in the treatment of neonatal jaundice: a comparative 804–809. study. J Paediatr Child Health 2010; 46: 234–237. 3 Seidman DS, Moise J, Ergaz Z, Laor A, Vreman HJ, Stevenson DK et al. Aprospective 22 Rosenfeld W, Sadhev S, Brunot V, Jhaveri R, Zabaleta I, Evans HE. Phototherapy effect randomized controlled study of phototherapy using blue and blue-green light-emitting on the incidence of patent ductus arteriosus in premature infants: prevention with devices, and conventional halogen-quartz phototherapy. J Perinatology 2003; 23: 123–127. chest shielding. Pediatrics 1986; 78: 10–14. 4 Tan KL. Comparison of the efficacy of fiberoptic and conventional phototherapy for 23 Travadi J, Simmer K, Ramsay J, Doherty D, Hagan R. Patent ductus arteriosus in neonatal hyperbilirubinemia. J Pediatr 1994; 125: 607–612. extremely preterm infants receiving phototherapy: does shielding the chest make a 5 Romagnoli C, Zecca E, Papacci P, Vento G, Girlando P, Latella C. Which phototherapy difference? A randomized, controlled trial. Acta Paediatr 2006; 95: 1418–1423. system is most effective in lowering serum bilirubin in very preterm infants? Fetal 24 Benders MJ, van Bel F, van de Bor M. Haemodynamic consequences of phototherapy in Diagn Ther 2006; 21: 204–209. term infants. Eur J Pediatr 1999; 158: 323–328. 6 Costello SA, Nyikal J, Yu VYH, McCloud P. Biliblanket phototherapy system versus 25 Benders MJ, van Bel F, van de Bor M. Cardiac output and ductal reopening during conventional phototherapy: a randomized controlled trial in preterm infants. phototherapy in preterm infants. Acta Paediatr 1999; 88: 1014–1019. J Paediatr Child Health 1995; 31: 11–13. 26 Pezzati M, Biagiotti R, Vangi V, Lombardi E, Wiechmann L, Rubaltelli FF. Changes in 7 Donzelli GP, Moroni M, Rapisardi G, Agati G, Fusi F. Fiberoptic phototherapy in the mesenteric blood flow response to feeding: Conventional versus fiber-optic management of jaundice in low birthweight infants. Acta Pediatr 1996; 85: 366–370. phototherapy. Pediatrics 2000; 105: 350–353. 8 Dani C, Martelli E, Reali MF, Bertini G, Panin G, Rubaltelli F. Fiberoptic and 27 Yao AC, Martinussen M, Johansen OJ, Brubakk AM. Phototherapy-associated changes conventional phototherapy effects on the skin of premature infants. J Pediatr 2001; in mesenteric blood flow response to feeding in term neonates. J Pediatr 1994; 124: 138(3): 438–440. 309–312.

Appendix Development (NICHD) provided grant support for the Neonatal Research Network’s (NRN’s) Phototherapy Trial Acknowledgements (2002 to 2005). The National Institutes of Health and the Eunice Kennedy Data collected at participating sites of the NICHD NRN were Shriver National Institute of Child Health and Human transmitted to RTI International, the data coordinating center

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(DCC) for the network, which stored, managed and analyzed the Stanford University Lucile Packard Children’s Hospital (U10 data for this study. On behalf of the NRN, Dr Abhik Das (DCC HD27880, M01 RR70)FSusan R Hintz, MD MS; M Bethany Ball, Principal Investigator), Dr Qing Yao, Dr Dennis Wallace and BS CCRC. Ms Sarah Kandefer (DCC Statisticians) had full access to all the University of Alabama at Birmingham Health System and data in the study and take responsibility for the integrity of the data Children’s Hospital of Alabama (U10 HD34216, M01 and accuracy of the data analysis. RR32)FWaldemar A Carlo, MD; Namasivayam Ambalavanan, We are indebted to our medical and nursing colleagues and the MD; Myriam Peralta-Carcelen, MD MPH; Monica V Collins, RN BSN infants and their parents who agreed to take part in this study. The MaEd; Shirley S Cosby, RN BSN; Vivien A Phillips, RN BSN. following investigators, in addition to those listed as authors, University of CaliforniaFSan Diego Medical Center and participated in this study: Sharp Mary Birch Hospital for Women (U10 HD40461)FNeil N NRN Steering Committee ChairsFAlan Jobe, MD PhD, Finer, MD; Yvonne E Vaucher, MD MPH; Maynard R Rasmussen University of Cincinnati (2001 to 2006); Michael S Caplan, MD, MD; David Kaegi, MD; Kathy Arnell, RNC; Clarence Demetrio, RN; University of Chicago, Pritzker School of Medicine (2006 to Martha G Fuller, RN MSN; Chris Henderson, RCP CRTT; Wade present). Rich, BSHS RRT. Alpert Medical School of Brown University and Women and University of Miami Holtz Children’s Hospital (U10 HD21397, Infants Hospital of Rhode Island (U10 HD27904)FAbbot R M01 RR16587)FCharles R Bauer, MD; Shahnaz Duara, MD; Laptook, MD; Angelita Hensman, BSN RNC; Lucy Noel RN. Silvia Hiriart-Fajardo, MD; Ruth Everett-Thomas, RN BSN; Amy Case Western Reserve University Rainbow Babies and Mur Worth, RN MS; Silvia Frade Eguaras, MS. Children’s Hospital (U10 HD21364, M01 RR80)FAvroy A University of Rochester Medical Center Golisano Children’s Fanaroff, MD; Michele C Walsh, MD MS; Deanne Wilson-Costello, Hospital (U10 HD40521, M01 RR44)FRonnie Guillet, MD PhD; MD; Nancy S Newman, RN; Bonnie S Siner, RN. Gary J Myers, MD; Linda J Reubens, RN CCRC; Diane Hust, MS RN Cincinnati Children’s Hospital Medical Center, University CS; Rosemary L Jensen; Erica Burnell, RN. Hospital and Good Samaritan Hospital (U10 HD27853, M01 University of Texas Southwestern Medical Center at Dallas RR8084)FEdward F Donovan, MD; Kurt Schibler, MD; Jean Parkland Health and Hospital System and Children’s Medical Steichen, MD; Barb Alexander, RN; Cathy Grisby, BSN CCRC; Center Dallas (U10 HD40689, M01 RR633)FWalid A Salhab, MD; Marcia Mersmann, RN; Holly Mincey, RN; Jody Shively, RN; Teresa Pablo J Sanchez, MD; Charles R Rosenfeld, MD; Roy J Heyne, MD; Gratton, PA. Jackie Hickman, RN; Gay Hensley, RN; Nancy A Miller, RN; Janet Duke University School of Medicine University Hospital, Morgan, RN. Alamance Regional Medical Center and Durham Regional Hospital University of Texas Health Science Center at Houston Medical (U10 HD40492, M01 RR30)FRonald N Goldberg, MD; C Michael School, Children’s Memorial Hermann Hospital and Lyndon Baines Cotten, MD MHS; Ricki F Goldstein, MD; Kathy J Auten, MSHS; Johnson General Hospital/Harris County Hospital District. Melody B Lohmeyer, RN MSN. (U10 HD21373, KL2 RR24149, UL1 RR24148)FKathleen Emory University Children’s Healthcare of Atlanta, Grady Kennedy, MD MPH; Pamela J Bradt, MD MPH; Patricia W Evans, Memorial Hospital and Emory University Hospital Midtown (U10 MD; Laura L Whiteley, MD; Esther G Akpa, RN BSN; Patty A Cluff, HD27851, M01 RR39)FBarbara J Stoll, MD; Ira Adams-Chapman, RN; Anna E Lis, RN BSN; Claudia I Franco, RNC MSN; Maegan MD; Ellen C Hale, RN BS CCRC. Currence, RN; Nora I. Alaniz, BS; Patti L Pierce Tate, RCP; Sharon Eunice Kennedy Shriver National Institute of Child Health and L Wright, MT(ASCP). Human DevelopmentFLinda L Wright, MD; Elizabeth M McClure, Wake Forest University Baptist Medical Center, Brenner MEd. Children’s Hospital, and Forsyth Medical Center (U10 HD40498, Indiana University Indiana University Hospital, Methodist M01 RR7122)FLisa K Washburn, MD; Nancy J Peters, RN CCRP; Hospital, Riley Hospital for Children and Wishard Health Services Barbara G Jackson, RN BSN. (U10 HD27856, M01 RR750)FBrenda B Poindexter, MD MS; Wayne State University Hutzel Women’s Hospital and Children’s James A Lemons, MD; Anna M Dusick, MD; Diana D Appel, RN Hospital of Michigan (U10 HD21385)FSeetha Shankaran, MD; Yvette BSN; Dianne E Herron, RN; Lucy C Miller, RN BSN CCRC; Leslie Johnson, MD; Athina Pappas, MD; Rebecca Bara, RN BSN; Geraldine Dawn Richard, BSN CCRC. Muran, RN BSN; Deborah Kennedy, RN BSN. RTI International (U10 HD36790)FW Kenneth Poole, PhD; Yale University Yale-New Haven Children’s Hospital (U10 Betty Hastings; Elizabeth N McClure, MEd; Jamie E Newman, PhD HD27871, M01 RR6022)FRichard A. Ehrenkranz, MD; Patricia MPH; Rebecca L Perritt, MS; Carolyn M Petrie Huitema, MS; Kristin Gettner, RN; Harris C Jacobs, MD; Christine Butler, MD; Patricia M Zaterka-Baxter, RN BSN. Cervone, RN; Monica Konstantino, RN BSN; Elaine Romano, MSN.

Journal of Perinatology Journal of Perinatology (2013) 33, 154–156 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp PERINATAL/NEONATAL CASE PRESENTATION Congenital chloride diarrhea presenting in newborn as a rare cause of meconium ileus

H Shamaly1,2, J Jamalia3, H Omari3, S Shalev2,4 and N Elias1,2 1Pediatric Department and Pediatric Pulmonology Unit, St Vincent Hospital, Nazareth, Israel; 2Department of Pediatrics, Bruce Rappaport School of Medicine, TechnionFInstitute of Technology, Haifa, Israel; 3Neonatal Intensive Care Unit. St Vincent French Hospital, Nazareth, Israel and 4Genetic Department, HaEmek Medical Center, Afula, Israel

polyhydramnios. Parents were not related, reported to be healthy Postpartum abdominal distention and meconium ileus may occur due to and with no family history of gastrointestinal disease. Physical intestinal obstruction, Hirschprung disease or cystic fibrosis. However, other examination revealed distended abdomen with no other significant rare and challenging etiologies such as congenital chloride diarrhea (CCD) findings. should be included in differential diagnosis of such presentation. We present Postnatally the infant did not pass meconium and abdominal a premature baby girl who had distended abdomen and lack of meconium distention increased significantly. Complete blood count, serum immediately after birth. Surgical etiology was excluded and she was electrolyte levels and kidney function tests were initially within mistakenly suspected of having cystic fibrosis due to meconium ileus. CCD normal limits. Abdominal X-ray revealed diffuse dilated intestinal was diagnosed by recognition of watery diarrhea in association with loops without signs of local obstructions (Figure 1). Barium enema hyponatremic, hypochloremic metabolic acidosis. Mutation analysis showed no signs of obstruction and excluded the diagnosis of confirmed the diagnosis. Hirschprung disease. The working clinical diagnosis at this point Journal of Perinatology (2013) 33, 154–156; doi:10.1038/jp.2012.42 was meconium ileus caused by cystic fibrosis. Treatment consisted Keywords: distended abdomen; meconium ileus; congenital chloride of repeated mucomyst (acetylcysteine) enemas along with gastric diarrhea decompression via nasogastric tube. After 5 days meconium passed, abdominal distention resolved and oral feeding was started. A few

Introduction Infants born with distended abdomen and absence of meconium passage require extensive investigation to arrive at an accurate diagnosis. Surgical causes such as small bowel obstructions, malrotation and Hirschprung disease should be excluded early to avoid signiﬁcant complications and high morbidity.1 Cystic ﬁbrosis is the main non-surgical etiology in newborns.2,3 However, other rare etiologies should be included in the differential diagnosis. We present a case of a premature newborn girl who was born with a distended abdomen and absence of meconium passage that was ultimately diagnosed as congenital chloride diarrhea (CCD).

Case A 36-week gestation female newborn of Arab descent was delivered by spontaneous vaginal delivery. Birth weight was 3015 g and the Apgar score was 9 and10 at 1 and 5 min, respectively. Prenatal ultrasound at 35 weeks gestation noted abdominal distention and

Correspondence: Dr N Elias, Pediatric Department and Pediatric Pulmonology Unit, St Vincent Hospital, PO Box 50294, Nazareth 16102, Israel. E-mail: [email protected] Received 17 January 2012; revised 22 February 2012; accepted 9 March 2012 Figure 1 Distended intestinal loops without signs of local obstruction. CCD a rare cause of meconium ileus in newborn H Shamaly et al 155

Table 1 Normal values at birth. Dehydration, hyponatremia, hypokalemia, hypochloremia at second week of life with normalization after NaCl and KCl solution started

Glucose (mg dl–1) Urea (mg dl–1) Creatinine (mg dl–1) Sodium (mmol l–1) Potassium (mmol l–1) Chloride (mmol l–1)

At birth 66 22 0.6 140 4.3 98 10 Days old 70 51 0.5 130 2.4 73 12 Days old 63 53 0.4 117 3.8 63 1 Week after NaCl and KCl treatment 88 23 0.5 138 5.1 92 days later, the baby developed hyponatremia, hypochloremia and patients and 2025–2026insATC in Polish patients. Other rare metabolic alkalosis. DNA genetic testing for mutations for cystic mutations were described as well in several other countries.11 fibrosis was negative. Urine chloride concentration was within In newborns presenting with distended abdomen and delayed normal limits. Watery diarrhea developed and increased fecal passage of meconium, both surgical causes and cystic fibrosis need chloride concentration (>100 mmol l–1; normal <40 mmol l–1) to be excluded. Co-existent metabolic disturbance such as was documented (Table 1). As repeated blood and fecal electrolytes hypochloremic and hypokalemic metabolic alkalosis should raise showed same results, CCD was considered as the presumed etiology. the suspicion of Barter disease as well. But clinicians should be The infant was treated with infusion of sodium chloride and vigilant to look for diarrhea in these newborns and include CCD in potassium solutions resulting ultimately in normal serum sodium differential diagnosis. As seen in our case, the diagnosis was only concentration. made at an age of 2 weeks and only after observing watery DNA sequencing revealed that the baby was homozygous for diarrhea. Obtaining fecal samples and measuring chloride C.559G >T disease-causing mutation in the exon 5 of SLC26A3 concentration is an easy and rapid way to make the diagnosis of gene, confirming the diagnosis of CCD. Both parents were found to CCD without delay. Making the correct diagnosis as soon as be heterozygous carriers of the same mutation. At 1 year of age, the possible is highly important as administration of early child was thriving and developing normally. treatment with NaCl and KCl solution supplements allows the maintenance of normal electrolyte and acid–base balance, normal growth and development, and favorable outcome of the disease.12 Discussion CCD is an autosomal recessive inherited disorder caused by a defect Conflict of interest in ClÀ/HCO exchange, mainly expressed in the apical brush 3 The authors declare no conflict of interest. border of ileal and colonic epithelium, resulting in chloride-rich diarrhea leading eventually to hypokalemia, hyponatremia and metabolic alkalosis.1,4 CCD may also present prenatally as Acknowledgments polyhydramnios due to intrauterine diarrhea leading to premature delivery.4–6 The disease is frequently found in Finland with We gratefully acknowledge the support of the Genetic Department of the University incidence of 1 in 30 0007 and in Arab children of Saudi Arabia and of Helsinki for genetic analysis of the patient and his parents. Kuwait with incidence of 1 in 5500.8,9 In spite of this relatively high incidence, the diagnosis is frequently delayed and confused with other similar diseases.6-8 References Intestinal obstruction of the newborn can be caused by multiple 1 Langer JC, Winthrop AL, Burrows RF, Issenman RM, Caco CC. False diagnosis of conditions.1 Surgical conditions include intestinal atresia, intestinal obstruction in a fetus with congenital chloride diarrhea. J Pediatr Surg 1991; malrotation, pyloric stenosis, annular pancreas, duplication cyst 26(11): 1282–1284. and Hirschprung disease. Common non-surgical etiologies include 2 Caspi B, Elchalal U, Lancet M, Chemke J. Prenatal diagnosis of cystic fibrosis: 6 ultrasonographic appearance of meconium ileus in the fetus. Prenat Diagn 1988; cystic fibrosis and Barter syndrome. The clinical signs and 8(5): 379–382. symptoms frequently overlap, making it difficult to distinguish 3 Efrati O, Nir J, Fraser D, Cohen-Cymberknoh M, Shoseyov D, Vilozni D et al. between them.7,8 Meconium ileus in patients with cystic fibrosis is not a risk factor for clinical Genetic testing for gene mutations causing CCD has become deterioration and survival: The Israeli Multicenter Study. J Pediatr Gastroenterol Nutr available to confirm the diagnosis. Our patient was found to have 2010; 50(2): 173–178. 4 Barss VA, Benacerraf BR, Frigoletto Jr FD. Antenatal sonographic diagnosis of fetal C.559G >T mutation similar to that reported in nine children gastrointestinal malformations. Pediatrics 1985; 76(3): 445–449. 9,10 from Kuwait and Saudi Arabia. Other frequent mutations were 5 Holmberg C. Congenital chloride diarrhoea. Clin Gastroenterol 1986; 15(3): described in the literature such as 951–953delGGT in Finish 583–602.

Journal of Perinatology CCD a rare cause of meconium ileus in newborn H Shamaly et al 156

6 Egritas O, Dalgic B, Wedenoja S. Congenital chloride diarrhea misdiagnosed as bartter 10 Hoglund P, Auranen M, Socha J, Popinska K, Nazer H, Rajaram U et al. Genetic syndrome. Turk J Gastroenterol 2011; 22(3): 321–323. background of congenital chloride diarrhea in high-incidence populations: Finland, 7 Kagalwalla AF. Congenital chloride diarrhea. a study in Arab children. J Clin Poland, and Saudi Arabia and Kuwait. Am J Hum Genet 1998; 63(3): 760–768. Gastroenterol 1994; 19(1): 36–40. 11 Kere J, Lohi H, Hoglund P. Genetic disorders of membrane transport III. Congenital 8 Al-Abbad A, Nazer H, Sanjad SA, Al-Sabban E. Congenital chloride diarrhea: a single chloride diarrhea. Am J Physiol 1999; 276(1 Pt 1): G7–G13. center experience with ten patients. Ann Saudi Med 1995; 15(5): 466–469. 12 Hihnala S, Hoglund P, Lammi L, Kokkonen J, Ormala T, Holmberg C. Long-term 9 Lundkvist K, Anneren G, Esscher T, Ewald U, Hardell LI. Surgical implications of clinical outcome in patients with congenital chloride diarrhea. J Pediatr Gastroenterol congenital chloride diarrhoea. Z Kinderchir 1983; 38(4): 217–219. Nutr 2006; 42(4): 369–375.

Journal of Perinatology Journal of Perinatology (2013) 33, 157–160 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp PERINATAL/NEONATAL CASE PRESENTATION Novel NKX2.1 mutation associated with hypothyroidism and lethal respiratory failure in a full-term neonate

ES Gillett1, GH Deutsch2, MJ Bamshad1, RM McAdams1 and PC Mann1 1Department of Pediatrics, Division of Neonatology, University of Washington School of Medicine, Seattle Children’s Hospital, Seattle, WA, USA and 2Department of Laboratories, University of Washington School of Medicine, Seattle Children’s Hospital, Seattle, WA, USA

Case We report a case of lethal neonatal hypoxic respiratory failure and A female infant was born by spontaneous vaginal delivery at hypothyroidism in an infant with a novel missense mutation in NKX2.1. 39 weeks and 5 days gestation to a 22-year-old Gravida 2 Para Journal of Perinatology (2013) 33, 157–160; doi:10.1038/jp.2012.50 1 female. The pregnancy was complicated by a positive Keywords: NKX2.1; hypothyroidism; respiratory failure; ECMO Group B streptococcus culture treated before delivery. At birth, meconium-stained amniotic fluid, poor respiratory effort and cyanosis were noted. Apgar scores were 5, 6 and 7 at 1, 5 and 10 min, respectively. The infant weighed 2839 g (9th percentile). Introduction A two-vessel cord was noted on physical examination. A chest radiograph demonstrated bilateral hazy lung fields consistent NKX2.1 encodes thyroid transcription factor-1, a nuclear with respiratory distress syndrome. Despite surfactant protein expressed in the brain, lung and thyroid during administration, respiratory status continued to worsen and bilateral embryonic development.1 Heterozygous mutations and deletions pneumothoraces occurred, necessitating support escalation to in NKX2.1 are associated with Brain-Lung-Thyroid Syndrome high-frequency oscillatory ventilation and inhaled nitric oxide. (BLTS, OMIM no. 610978), whose features include congenital Hypotension developed requiring pressor support with inotropic hypothyroidism, pulmonary dysfunction and neurological drips, and echocardiogram revealed severe right ventricular impairment with variable phenotypic severity.2–5 We report dysfunction. Given persistent hypoxemic respiratory failure, the a novel missense mutation in NKX2.1 identified in an infant infant was transferred to our tertiary care facility on day of with respiratory failure and hypothyroidism in the immediate age 1 and placed on venoarterial extracorporeal membrane postnatal period. oxygenation (ECMO). Following placement on ECMO, inotropic support was no longer required. Newborn screening revealed an elevated thyroid-stimulating Methods hormone of 180.7 mIU mlÀ1 and levothyroxine replacement was Immunohistochemistry was performed on sequential, formalin- started. Hypothyroidism was confirmed at 1 week of life with a À1 fixed, paraffin-embedded, 5-mm lung sections on a Ventana thyroid-stimulating hormone of 93.7 mIU ml and low, normal, À1 automated immunostainer (Ventana Medical Systems, Tucson, AZ, free thyroxine (1.1 ng dl ). Initial blood cultures remained USA) after antigen retrieval, using the following antibodies: thyroid without growth. Throughout hospitalization, the infant exhibited transcription factor-1 (1:50) and thyroglobulin (1:100; both mouse unremitting respiratory failure in the setting of significant monoclonal; Thermo Scientific, Rockford, IL, USA), surfactant pulmonary hypertension with the right heart failure by proteins (SP)-A (1:4000), SP-B (1:2000) and proSP-C (1:2000; echocardiography. Following multiple unsuccessful attempts all rabbit polyclonal; Chemicon, Temecula, CA, USA), CC10/CCSP to support the infant off ECMO, the decision was made to (1:1000, rabbit polyclonal; Santa Cruz Biotechnology Inc, withdraw care on day of age 28. Santa Cruz, CA), ABCA3 (1:1000, rabbit polyclonal; Seven Hills The combination of neonatal respiratory failure and Bioreagents, Cincinnati, OH, USA). hypothyroidism led to clinical suspicion of BLTS and sequencing of NKX2.1. Sequencing by Nemours Molecular Diagnostics Correspondence: Dr PC Mann, Departments of Pediatrics, Division of Neonatology, University Laboratory (Wilmington, DE, USA) revealed a novel heterozygous of Washington, Seattle Children’s Hospital, 4800 Sand Point Way NE, Seattle, WA 98105, USA. E-mail: [email protected] missense mutation in exon 3 of NKX2.1, c.621C > G with Received 1 December 2011; revised 20 March 2012; accepted 26 March 2012 resultant amino acid substitution p.Ile207Met. No mutations were Novel NKX2.1 mutation in neonate ES Gillett et al 158 detected in SFTPB or SFTPC, which encode SP, or ABCA3, which is pneumocytes (Figure 1c), but intact SP-B, SP-A, ABCA3 and critical for surfactant metabolism.6 Although the infant’s parents Clara cell secretory protein. Compared with age-matched controls, elected not to undergo genetic testing themselves, family histories there was no detectable difference in the pattern or intensity of respiratory impairment and thyroid disease were denied. of NKX2.1 staining (Figure 1d). By ultrastructural analysis, At autopsy, the lungs were severely congested (combined lung numerous well-formed lamellar bodies were present within type II weight 121.7 g, expected 60 g) with diffuse alteration of the pneumocytes, which comprised the majority (>75%) of the architecture by alveolar remodeling and Type II cell hyperplasia. alveolar epithelial cells. The heart (23.1 g, expected 20 g) had a There was alveolar filling by granular proteinaceous material, patent ductus arteriosus with no ischemic or hypertrophic change.8 focally periodic acid-Schiff-positive, diastase resistant, as well The thyroid gland was symmetrical and appeared appropriate size as macrophages, edema and fibrin (Figure 1b). The radial for age. Microscopic examination showed colloid depletion, but alveolar count was markedly reduced (average 2, expected >5)7 normally formed follicles and follicular epithelium (Figure 2a); (Figure 1a). Immunohistochemical staining for SP was expression levels of NKX2.1 (Figure 2b) and thyroglobulin remarkable for near-complete absence of proSP-C within type II were comparable to controls. The brain weight was reduced

Figure 1 Lung histology and immunohistochemistry. (a) Diffuse alteration of lung architecture with deﬁcient lung growth evident by close proximity of bronchioles (*) to pleural surface (arrow; hematoxylin and eosin, Â 40). (b) Alveolar proteinosis with reactive type II pneumocytes reﬂective of altered surfactant metabolism (periodic acid-Schiff diastase, Â 200). (c) ProSP-C immunoreactivity limited to rare type II pneumocytes (arrow) compared with age-matched control (insert; Â 200). (d) Nuclear NKX2.1 protein within epithelium and type II pneumocytes, similar to control (insert; Â 200).

Figure 2 Thyroid histology and immunohistochemistry. (a) Postmortem thyroid follicular architecture (hematoxylin and eosin, Â 200). (b) Nuclear NKX2.1 staining of follicular epithelial cells ( Â 200).

Journal of Perinatology Novel NKX2.1 mutation in neonate ES Gillett et al 159

(349 g, expected 413 g) with gross and microscopic appearance until after the infant expired off ECMO. This clinical presentation indistinguishable from the changes seen with hypoxic ischemic was consistent with only a limited number of genetic diagnoses. encephalopathy.8 The targeted sequencing of NKX2.1 and screening of other candidate genes to exclude alternative diagnoses represents a current state-of-the-art molecular diagnostic approach. A list of Discussion laboratories that test for NKX2.1 mutations is available at We report a second case of lethal neonatal respiratory distress genetests.org.17 at birth in an infant with BLTS.9 We also discovered a novel In summary, we report the clinical characteristics of an infant heterogyzous mutation in NKX2.1 predicted to encode an who died from severe pulmonary insufficiency caused by a novel p.Ile207Met amino acid substitution as causative for this BLTS mutation in NKX2.1. This is the second reported case of a neonatal case. This mutation maps to the DNA-binding domain of NKX2.1.9 death from respiratory failure due to a heterozygous NKX2.1 Although the parents refused genetic testing, most BLTS cases result mutation. Screening of NKX2.1 should be considered in all from spontaneous mutation. A different substitution of this same neonates who present with respiratory distress and hypothyroidism amino acid residue, p.Ile207Phe, has been reported previously by at birth. Maquet et al.9 in an infant with a similar clinical course. Together, these observations suggest that disruption of this amino acid residue might be predictive of poor outcome due to severe Conflict of interest pulmonary dysfunction. This hypothesis is supported by functional The authors declare no conflict of interest. studies demonstrating that p.Ile207Phe substitution results in abnormal DNA binding and transactivation of the thyroglobulin and SP-B genes.9 References Described lung pathology in individuals with NKX2.1 mutations 1 Lazzaro D, Price M, de Felice M, Di Lauro R. The transcription factor TTF-1 is 9–12 is limited and varied. Although the predominant histological expressed at the onset of thyroid and lung morphogenesis and in restricted regions of feature is altered surfactant metabolism with pulmonary the foetal brain. Development 1991; 113: 1093–1104. alveolar proteinosis, other cases have demonstrated deficient 2 Devriendt K, Vanhole C, Matthijs G, de Zegher F. Deletion of thyroid transcription lung growth as the principal defect.10 Pulmonary alveolar factor-1 gene in an infant with neonatal thyroid dysfunction and respiratory failure. N Engl J Med 1998; 338: 1317–1318. proteinosis, significant pulmonary hypoplasia and a striking 3 Krude H, Schutz B, Biebermann H, von Moers A, Schnabel D, Neitzel H et al. reduction in proSP-C expression were all present in our case, Choreoathetosis, hypothyroidism, and pulmonary alterations due to human NKX2-1 as well as superimposed diffuse alveolar damage. Unlike the haploinsufficiency. J Clin Invest 2002; 109: 475–480. report by Maquet et al.,9 SP-B expression was intact. Altered 4 Iwatani N, Mabe H, Devriendt K, Kodama M, Miike T. Deletion of NKX2.1 gene SP expression has been seen in some NKX2.1 mutations,9–11 encoding thyroid transcription factor-1 in two siblings with hypothyroidism and and NKX2.1 has known regulatory effects on the SP-C gene respiratory failure. J Pediatr 2000; 137: 272–276. 13 5 Kleinlein B, Griese M, Liebisch G, Krude H, Lohse P, Aslanidis C et al. Fatal neonatal expression. Reports of thyroid pathology in individuals with respiratory failure in an infant with congenital hypothyroidism due to NKX2.1 mutations are variable and range from normal haploinsufficiency of the NKX2-1 gene: alteration of pulmonary surfactant architecture to hemiagenesis.9–11 Individuals with NKX2.1 homeostasis. Arch Dis Child Fetal Neonatal Ed 2011; 96: F453–F456. mutations frequently have clinical and pathological 6 Schulenin S, Nogee LM, Annilo T, Wert SE, Whitsett JA, Dean M. ABCA3 gene mutations neurological brain alterations,14 but no changes beyond in newborns with fatal surfactant deficiency. N Engl J Med 2004; 350: 1296–1303. 7 Conney TP, Thurlbeck WM. The radial alveolar count method of Emery and Mithal: those associated with hypoxic ischemic encephalopathy were a reappraisal 1Fpostnatal lung growth. Thorax 1982; 37: 572–579. evident. 8 Siebert JR. Perinatal, fetal and embryonic autopsy. In: Gilbert-Barness E, Kapur RP, This case raised questions about the appropriateness and timing Oligny LL, Siebert JR (eds). Potter’s Pathology of the Fetus, Infant and Child 2nd edn. of lung biopsy in neonates showing minimal clinical improvement Mosby Elsevier: Philadelphia, 2007, pp 725. on ECMO. Although limited empirical data are available to guide 9 Maquet E, Costagliola S, Parma J, Christophe-Hobertus C, Oligny LL, Fournet JC et al. biopsy timing, a retrospective analysis of 173 neonates with acute Lethal respiratory failure and mild primary hypothyroidism in a term girl with a de novo heterozygous mutation in the TITF1/NKX2.1 gene. J Clin Endocrinol Metab hypoxemic respiratory failure treated with ECMO recommended 2009; 94: 197–203. considering lung biopsy after 7 to 10 days in infants with 10 Galambos C, Levy H, Cannon CL, Vargas SO, Reid LM, Cleveland R et al. Pulmonary symptoms of alveolar capillary dysplasia.15 Open-lung biopsy is pathology in thyroid transcription factor-1 deficiency syndrome. Am J Respir Crit Care safe in patients on ECMO16 and might have shortened ECMO Med 2010; 182: 549–554. duration in this case once lung pathology became apparent. 11 Guillot L, Carre A, Szinnai G, Castanet M, Tron E, Jaubert F et al. NKX2-1 mutations leading to surfactant protein promoter dysregulation cause interstitial lung disease Earlier access to diagnostic genetic testing results might also in ‘‘Brain-Lung-Thyroid Syndrome’’. Hum Mutat 2010; 31: E1146–E1162. have facilitated decision-making about the level and length of 12 Willemsen MA, Breedveld GJ, Wouda S, Otten BJ, Yntema JL, Lammens M et al. supportive care provided. Genetic testing results were not available Brain-Thyroid-Lung syndrome: a patient with a severe multi-system disorder due to

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a de novo mutation in the thyroid transcription factor 1 gene. Eur J Pediatr 2005; 15 Cassidy J, Smith J, Goldman A, Haynes S, Smith E, Wright C et al. The incidence and 164: 28–30. characteristics of neonatal irreversible lung dysplasia. J Pediatr 2002; 141: 426–428. 13 Liu C, Glasser SW, Wan H, Whitsett JA. GATA-6 and thyroid transcription factor-1 16 Inwald D, Brown K, Gensini F, Malone M, Goldman A. Open lung biopsy in neonatal directly interact and regulate surfactant protein-C gene expression. J Biol Chem 2002; and paediatric patients referred for extracorporeal membrane oxygenation (ECMO). 277: 4519–4525. Thorax 2004; 59: 328–333. 14 Carre A, Szinnai G, Castanet M, Sura-Trueba S, Tron E, Broutin-L’Hermite I et al. 17 Gene Tests: Medical Genetics Information Resource (database online). Five new TTF1/NKX2.1 mutations in brain-lung-thyroid syndrome: rescue by PAX8 Copyright: University of Washington, Seattle, WA, USA. 1993–2012. Available at synergism in one case. Hum Mol Genet 2009; 18: 2266–2276. http://www.genetests.org, Accessed 9 February 2012.

Journal of Perinatology Journal of Perinatology (2013) 33, 141–146 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Oral L-arginine supplementation and faecal calprotectin levels in very low birth weight neonates

E Polycarpou1, S Zachaki2,3, V Papaevangelou4, M Tsolia4, A Kyriacou5, C Kostalos1 and D Kafetzis4 1Neonatal Intensive Care Unit, ‘Alexandra’ Hospital, Athens, Greece; 2Health Physics and Environmental Health Laboratory, NCSR ‘Demokritos’, Athens, Greece; 3Department of Medical Genetics, Athens University, St Sophia’s Children’s Hospital, Athens, Greece; 4Second Department of Paediatrics, Athens University, Children’s Hospital ‘Aglaia Kyriakou’, Athens, Greece and 5Department of Nutrition and Dietetics, Harokopion University, Athens, Greece

NEC is the most common acquired gastrointestinal disease in Objective: The objective of this study is to determine the potential effect of premature infants. In NEC, the small (most often distal) and/or oral L-arginine supplementation on intestinal inflammation in very low large bowel becomes injured, develops intramural air, and this may birth weight (VLBW) neonates, as estimated by faecal calprotectin levels. progress to frank necrosis with perforation.5 The high mortality Study Design: The study enrolled 83 VLBW neonates with birth weight and morbidity from NEC have not significantly improved during 5,6 p1500 g and gestational age p34 weeks. In this double-blind study, the last 40 years. Although extensive research has investigated 40 neonates received daily oral L-arginine supplementation of the pathophysiology of NEC, a complete understanding has not 1.5 mmol kg1 per day between the 3rd and 28th day of life, and 43 been fully elucidated. neonates placebo. Stool samples were collected on days 3, 14 and 28, and The pathology of NEC frequently resembles intestinal ischaemia calprotectin was measured by enzyme-linked immunosorbent assay. reperfusion injury. An important regulator of vascular perfusion is endothelial nitric oxide (NO), an anti-inflammatory Result: Calprotectin values significantly decreased over time in both chemical mediator and vasodilator involved in the maintenance groups (P ¼ 0.032). No difference in faecal calprotectin values was of mucosal integrity, intestinal barrier function and regulation recorded between neonates receiving arginine supplementation and of intestinal mucosal flow in the face of inflammation or neonates receiving placebo at days 3, 14 and 28. injury.7,8 Inhibition of NO synthesis in a variety of animal Conclusion: Faecal calprotectin values decrease with increasing models, in which bowel injury is induced, increases the area of postnatal age in VLBW infants, but this is not related to arginine intestinal damage.7,8 NO is synthetised from the amino acid supplementation. L-arginine by NO synthases. Journal of Perinatology (2013) 33, 141–146; doi:10.1038/jp.2012.51; Arginine is a ‘conditionally’ essential amino acid, meaning that published online 3 May 2012 endogenous arginine production covers metabolic requirements in healthy, unstressed individuals, but becomes an essential amino Keywords: arginine; calprotectin; necrotising enterocolitis; nitric oxide acid under conditions of increased need, for example, growth or tissue repair, or in catabolic states, such as sepsis and starvation.9 Introduction In prematurely born human neonates, hypoargininaemia is frequently observed10 and hypothesised to predispose such infants Calprotectin is a 36.5-kDa calcium and zinc-binding protein that to the development of NEC.11–13 A relative arginine deficiency or constitutes about 60% of all soluble cytosol proteins in human immaturity of NO synthases activity in premature infants may lead neutrophil granulocytes.1 Its faecal measurement has been to deficient tissue NO levels, vasoconstriction and ischaemia recognised as a reliable marker for the detection of gastrointestinal reperfusion injury, and may predispose to NEC. Plasma arginine tract inflammatory activity in both children and adults. It can be a levels were found to be decreased in premature infants with useful biomarker of gastrointestinal mucosa inflammation, and NEC,14–16 whereas other studies suggest that arginine thus, of necrotising enterocolitis (NEC) in premature infants.2–4 supplementation can reduce the incidence of NEC.17,18 Correspondence: Dr E Polycarpou, Neonatal Intensive Care Unit, ‘‘Alexandra’’ Hospital, 81 The aim of this double blind study was to determine whether Holborn House, Du cane road, London W12 0TS, UK. arginine supplementation in very low birth weight (VLBW) E-mail: [email protected] Received 6 January 2012; revised 19 March 2012; accepted 2 April 2012; published online 3 May neonates reduces gut inflammation, as assessed by faecal 2012 calprotectin concentrations. Faecal calprotectin in neonates receiving arginine E Polycarpou et al 142

Methods a stadiometer to measure body length, and a non-stretchable tape Neonates to assess head circumference. The present study enrolled 83 VLBW neonates with birth weight p1500 g and gestational age p34 weeks, as determined by the Faecal calprotectin levels last maternal menstrual period and Ballard scoring system,19 Sample collection, sample preparation and analysis. Stool consecutively born in ‘Alexandra’ Hospital in Athens, Greece, between samples were collected from the neonates’ diapers with sterile June 2009 and September 2010. Infants were excluded if they had plastic spoons and put in sterile plastic screw-cap tubes at 3rd, 14th lethal congenital or chromosomal abnormalities, inborn errors of and 28th day after birth. All stool samples were stored at 4 1C, and metabolism, or if the parents did not give consent. The study faecal calprotectin levels were measured within 3 days using protocol was approved by the Ethics Committee of the Hospital, and Calprotectin ELISA KIT (Bu¨hlmann Laboratories AG, Scho¨nenbuch, all parents signed informed consent before enrolment. Switzerland), according to the manufacturer’s instructions. In detail, after the sample collection and a short extraction procedure Study protocol (1 g stool sample: 4.9 ml extraction buffer), the extract was stored In this double-blind study, 83 VLBW neonates were prospectively at 20 1C until the measurement of calprotectin. A sample randomly assigned either to receive a daily oral L-arginine dilution of 1:50 was applied for all samples, which gives accurate supplement of 1.5 mmol kg1 per day (261 mg kg1) suspended in measurements for calprotectin values within the range of 10 to water for injection in two equal doses with oral feeds, from the 3rd 600 mgg1 (lower range procedure). For samples exceeding to the 28th day after birth (N ¼ 40; arginine group), or placebo 600 mgg1, a sample dilution of 1:150 was also applied, which containing 5% glucose in equivalent volume (N ¼ 43; control gives more accurate measurements for calprotectin values within group). The pharmacy prepared dilutions of the same volume of the range 600 to 1800 mgg1 (extended range procedure). All the oral arginine supplement and placebo containing 5% glucose, controls and specimen was measured in duplicate. and the nurses giving the dilutions were not aware of the diluent Incidence of NEC in the first 3 months of life: diagnosis and constitute (not transparent bottles) and were blinded to the classification of NEC. The diagnosis and classification of NEC was allocation of patients. done according to modified Bells’ criteria.22–24 The neonatologists who The dose of arginine was based on previous studies.18 All babies classified the episodes of NEC, as well as the radiologist who evaluated were observed for side effects, such as diarrhoea, vomiting, low the abdominal X-rays were blinded to the allocation of patients. blood pressure and hypoglycaemia/hyperglycaemia. Sample size. We calculated that a sample size of 45 neonates in Enteral feeds were commenced at 10 to 25 ml kg1 per day on each group will have 80% power to detect a difference in means of the first day of life (minimal feeding) and increased on the fourth 60 mgg1 in calprotectin concentrations, assuming that the day of life, and forward by 10 to 25 ml kg1 per day as tolerated, common s.d. between the two groups is 100, using a two group up to a maximum of 150 to 180 ml kg1 per day. Parenteral t-test with a 0.05 two-sided significance level. The study was not amino acids (Vamin 6.5%) was started on the first day of life at a priori powered to detect differences in NEC incidence. 1 1 1.5 g kg per day and gradually increased to 3.5 g kg per day. 2 Vamin 6.5% contains 4.1 g of L-arginine per 1000 ml. Breast milk Statistical analysis. Pearson’s w -test with continuity correction 20 contains about 54 mg of L-arginine per 100 ml, although the of Fisher’s exact test were used to investigate the association actual arginine content of own mother’s milk was not measured. between demographic/clinical data and the groups of interest Breast milk was always fortified (Nutricia, Amsterdam, Netherlands) (arginine vs control group). Day 3 values regarding faecal with two sachets of fortified breast milk/100 ml. Full-strength calprotectin, head growth and weight were tested between arginine breast milk fortification (two sachets in 100 ml of milk) increases and control group with independent sample t-test. Changes over the concentration of L-arginine in the milk by 24 mg per 100 ml. time between arginine and control group were examined as in Thus, breast-fed infants received about 78 mg per 100 ml repeated-measures analysis mixed-effects model assigning a arginine from milk. Formula-fed infants received Almiron random intercept for each subject. premature (Almiron premature, Numico, Amsterdam, To reduce the sample variability, faecal calprotectin was Netherlands), which contains 82.6 mg L-arginine per 100 ml, a transformed into the respective natural logarithmic scale. Sample concentration in the recommended range of arginine in preterm descriptives regarding these variables are presented by geometric formula.21 No bank milk (donors’ milk) was used. means. Significance level is set at 5%. Statistical package SAS V9.2 (SAS Institute, Cary, NC, USA) has been used for the data analysis. Clinical data Demographic data, fluid intake, total caloric intake, total arginine Results intake, total protein intake, weight and head growth were recorded. Between June 2009 and September 2010, 171 neonates of p34 and We used an electronic scale with accuracy of 10 g to weigh babies, birth weight p1500 g were born in ‘Alexandra’ University

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Maternity Hospital. Of these, 19 (11%) died in the first few days steroids. No neonate had persistent hypoglycaemia. No adverse of life, 4 were excluded from the study (1 was diagnosed with effects, such as diarrhoea, vomiting, low blood pressure and phenylketonuria, 2 had known chromosomal abnormalities and hypoglycaemia/hyperglycaemia were observed in neonates 1 had severe congenital anomalies), 33 neonates had to be receiving arginine supplementation. transferred to other Units in the first days after birth, because of no In Table 2, the incidence of common events occurring in cot availability. Parents of 32 neonates declined consent. The preterm neonates is shown. No statistically significant differences remaining 83 neonates were enrolled in the study. between arginine and control groups were observed. The demographic data of the neonates receiving L-arginine Table 3 describes the incidence, morbidity and mortality supplementation (arginine group, N ¼ 40) and neonates receiving associated with NEC in the two groups. placebo (control group, N ¼ 43) are shown in Table 1. No Neonates in both arginine and control group had a significant significant differences were noted. No neonate received postnatal increase in weight and head circumference over time (P<0.0001).

Table 1 Demographic data of neonates receiving oral arginine supplementation (N ¼ 40) and controls (N ¼ 43)

Neonates receiving arginine, Neonates not receiving arginine, Statistical signiﬁcance N ¼ 40 (%) N ¼ 43 (%) difference

Sex Male (%) 17 (42.5) 19 (44.2) n.s. Female (%) 23 (57.5) 24 (55.8) Weight (g)a 1168 (1095.1, 1242.2) 1127 (1047.1, 1207.6) n.s. Gestational age (weeks)a 29.2 (28.9, 29.4) 28.8 (28.5–29.1) n.s. Singleton (%) 24 (60) 30 (70) n.s. Twins-triplets (%) 16 (40) 14 (30) Caesarean section (%) 30 (75) 32 (74.4) n.s. Vaginal delivery (%) 10 (25) 11 (25.6) Breast milk (%) 7 (17.5) 5 (11.6) n.s. Preterm formula (%) 33 (82.5) 38 (88.4)

Apgar score 1 minb 7 7 n.s. 5 minb 8 8 n.s.

IUGR (%) 16 (40) 14 (32.6) n.s. Antenatal steroids (%) 32 (80) 34 (79.1) n.s. Maternal antibiotics during labour (%) 14 (35) 18 (42) n.s.

Abbreviation: n.s., no statistical signiﬁcance. aMean value (±s.d.). bMedian value.

Table 2 Incidence of common events occurring in preterm neonates in the two groups of neonates

Neonates receiving arginine, Neonates not receiving arginine, Statistical signiﬁcance N ¼ 40 (%) N ¼ 43 (%) difference

RDS (%) 31 (77.5) 35 (81.4) n.s. PDA (%) 6 (15) 8 (18.6) n.s. PDA treated with ibuprofen (%) 4 (10) 7 (16.3) n.s. IVH Grade III and IV (%) 9 (22.5) 12 (27.9) n.s. Umbilical arterial catheter (%) 26 (60.5) 31 (72.1) n.s. Days of umbilical arterial cathetera 7 5.5 n.s.

Abbreviations: IVH, intraventricular haemorrhage; n.s., no statistical signiﬁcance; PDA, patent ductus arteriosis; RDS, respiratory distress syndrome. aMedian value.

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Changes over time between arginine and control group concerning difference in the mean calprotectin values between the weight gain and head growth were similar in both groups two groups on day 3, 14 and 28 (P ¼ 0.60, P ¼ 0.18 and (P ¼ 0.43 for weight gain and P ¼ 0.31 for head growth). Values P ¼ 0.72, respectively). Moreover, comparing the calprotectin are presented in Table 4. values over time within the groups, we found that these values Mean calprotectin values on day 3, 14 and 28 were in the significantly decreased over time in both groups (P ¼ 0.032). arginine group 269.9 (187.5 to 388.4), 166.8 (109.3 to 254.6) and This decrease over time was similar in both groups (P ¼ 0.554; 112.74 (71.1 to 178.8) mgg1 versus 237.8 (170.7 to 331.3), 111 Figure 1). (71.6 to 172) and 126.1 (82.2 to 193.5) mgg1 in the control There were no differences in fluid or caloric intake between group, respectively (Table 5). There was no statistically significant neonates receiving L-arginine supplementation and neonates

Table 3 Comparison of incidence, morbidity and mortality due to NEC in the two groups

Neonates receiving arginine, Neonates not receiving arginine, Statistical signiﬁcance N ¼ 40 (%) N ¼ 43 (%) difference

Incidence of NEC (stages II and III) 4 (10%) 10 (23.3%) n.s. Age of NEC diagnosis (days)a 17 15 n.s. Surgery for NEC 0 (0%) 2 (4.7%) n.s. Incidence of death due to NEC 0 (0%) 4 (9.3%) P ¼ 0.045 Incidence of death due to other causes 8 (20%) 8 (18.6%) n.s.

Abbreviations: NEC, necrotizing enterocolitis; n.s., no statistical signiﬁcance. aMedian.

Table 4 Growth, protein, arginine, ﬂuid and caloric intake in arginine and placebo groups on days 3, 14 and 28 after birth

Day 3 Day 14 Day 28

Arginine group Placebo group Arginine group Placebo group Arginine group Placebo group

Weight (g) 1115 (1039.5, 1190.3) 1066 (992, 1140.4) 1251 (1167.2, 1335.6) 1197 (1105.1, 1288) 1549 (1430.4, 1666.7) 1458 (1352.1, 1563.7) Head circumference (cm) 26.9 (26.2, 27.5) 26.7 (26.1, 27.2) 27.9 (27.4, 28.5) 27.3 (26.6, 28) 29.7 (28.9, 30.5) 29.3 (28.5, 30.1) Total ﬂuid intake (ml kg1 per day) 121 126 171 170 184 176 Total enteral feeds (ml kg1 per day) 17.1 14.0 85.7 61.5 160.9 139.4 Urea (mg dl1) 404441342121 Total arginine intake (mg kg1 per day)a 409.4 158.3 453.7 219.3 432 171.9 Total arginine intake (mmol kg1 per day) 2.35 0.91 2.6 1.26 2.5 0.99 Total caloric intake (Kcal kg1 per day) 61.6 60.7 111.9 104.3 142 130.4

All the values expressed as mean (95% conference interval). aArginine group received 1.5 mmol kg1 per day arginine supplementation.

Table 5 Comparison of faecal calprotectin values on days 3, 14 and 28

Faecal calprotectin values (mgg1) Statistical signiﬁcant difference

Day 3 Day 14 Day 28

Total population (N ¼ 83) 252.7 (198.7–321.4) 119.4 (88.0–162.2) 133.4 (98.5–180.7) Arginine group (N ¼ 40) 269.9 (187.5–388.4) 166.8 (109.3–254.6) 112.7 (71.1–178.8) n.s. Placebo group (N ¼ 43) 237.8 (170.7–331.3) 111.0 (71.6–172.0) 126.1 (82.2–193.5) Fortiﬁed breast milk (N ¼ 12) 235.7 (102.1–544.1) 128.4 (69.0–238.9) 98.4 (45.9–201.0) n.s. Formula (N ¼ 71) 255.9 (198.8–329.4) 134.3 (95.1–189.8) 124.1 (88.0–175.0)

Abbreviations: n.s., no statistical signiﬁcance. All values are expressed as means (95% conﬁdence intervals).

Journal of Perinatology Faecal calprotectin in neonates receiving arginine E Polycarpou et al 145

and 28. Possible explanations of this could be that calprotectin is not a reliable biomarker for early diagnosis of bowel inflammation in neonates, or that arginine supplementation in the dose used has no effect on bowel inflammation. The target difference between the two groups set in this study was 60 mgg1. With a much larger sample, even a very small difference such the one found on day 28 (20 mgg1), if it exists, could be proven to be statistically significant. We would recommend larger studies with different doses of arginine and/or longer duration of treatment. Interestingly, the mean calprotectin value is lower on day 28 in breast-milk-fed neonates receiving arginine supplementation, comparing with neonates being fed with formula (with and Figure 1 Comparison of mean calprotectin values (mgg1) at days 3, 14 and without arginine supplementation) and neonates receiving breast 28 in arginine-supplemented and control group. milk without arginine supplementation, but not significantly (Table 5). There may be a synergistic preventive action of arginine receiving placebo on days 3, 14 and 28. There is a significant with breast milk, and it would be interesting to investigate it in a difference in arginine intake due to arginine supplementation. larger study with babies receiving breast milk with or without arginine supplementation. The incidence of NEC (stage II and III) in our study was found Discussion to be higher in the control group comparing with the arginine In this randomised double-blind trial, our aim was to determine group (23.3% vs 10%, respectively), but this does not reach whether arginine supplementation leads to lower calprotectin statistical significance. A sample size of minimum 122 neonates values, indicating that neonates receiving arginine per group would be required to detect a reduction in the incidence supplementation have a milder inflammatory response. of NEC from 23.3 to 10% with a two-sided 5% significance level and Arginine supplementation was administered exclusively orally, a power of 80%. considering that arginine could have beneficial effect locally on the In conclusion, faecal calprotectin values decrease with intestinal mucosa itself, increasing proliferation and differentiation increasing postnatal age in VLBW infants, but this does not seem to because of the production of other amino acids and polyamines.9,25 be related to arginine supplementation. Thus, either arginine has No adverse effects from oral arginine supplementation of no influence on bowel inflammation in the dose used or faecal 1.5 mmol kg1 per day were noted. Considerably higher calprotectin is not a good biomarker for early stages of therapeutic doses of L-arginine have been used (loading inflammation. 600 mg kg1 followed by an infusion of 250 mg kg1 per day) without adverse effects in the management of urea cycle disorders.26,27 Conflict of interest The usefulness of faecal calprotectin as a marker of bowel The authors declare no conflict of interest. inflammation remains controversial in the neonatal period because of high levels in this age range. The reference normal upper value for healthy adults and children ages from 4 to 17 years, regardless Acknowledgments of sex, is 50 mgg1.28 Several groups have reported that term and preterm infants have a wide range and higher average faecal We thank all the parents for their willingness to participate in this research study calprotectin levels than healthy adults.29–34 Calprotectin values of and all the nurses of the Neonatal Unit of ‘Alexandra’ Hospital for their help with the study. Further, we acknowledge EM Delicha for statistical analysis and NP 363 and 636 mgg1 have been recommended as a cut-off for the Polycarpou for the evaluation of X-rays. This work is registered in detection of mild or severe enteropathy in preterm neonates, ClinicalTrials.gov with Unique Protocol ID: ALEXANDRA 9159. respectively.31 A high average calprotectin value was found on day 3 (meconium before arginine supplementation) in both groups. This References is consistent with previous studies measuring calprotectin levels in meconium in VLBW infants.35 In agreement with others,31,33 1 Dale I, Fagerhol MK, Naesgaard I. Purification and partial characterization of a highly immunogenic human leukocyte protein, the L1 antigen. Eur J Biochem 1983; 134: we found that faecal calprotectin decreases with postnatal age. 1–6. There was no significant difference comparing the mean 2 Carroll D, Corfield A, Spicer R, Cairns P. Faecal calprotectin concentrations and calprotectin values in the arginine and control group on day 3, 14 diagnosis of necrotising enterocolitis. Lancet 2003; 361: 310–311.

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3 Vieten D, Cairns P. The role of calprotectin in the diagnosis of neonatal necrotizing 20 Lemons JA, Reyman D, Moye L. Amino acid composition of preterm and term breast enterocolitis. Ir Med J 2005; 98: 69. milk during early lactation. Early Hum Dev 1983; 8: 323–329. 4 Thuijls G, Derikx JP, van Wijck K, Zimmermann LJ, Degraeuwe PL, Mulder TL et al. 21 Klein CJ. Nutrient requirements for preterm infant formulas. J Nutr 2002; Non-invasive markers for early diagnosis and determination of the severity of 132(6 Suppl 1): 1395S–13577. necrotizing enterocolitis. Ann Surg 2010; 251: 1174–1180. 22 Bell MJ, Ternberg JL, Feigin RD, Keating JP, Marshall R, Barton L et al. Neonatal 5 Srinivasan PS, Brandler MD, D’Souza A. Necrotizing Enterocolitis. Clin Perinatol 2008; necrotising enterocolitis. Therapeutic decisions based upon clinical staging. Ann Surg 35: 251–272. 1978; 187:1–7. 6 de Oliveira ND, Miyoshi MH. Advances in necrotizing enterocolitis. J Pediatr (Rio J) 23 Neu J. Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to 2005; 81(1suppl): S16–S22. prevention. Pediatr Clin North Am 1996; 43: 409–432. 7 MacKendrick W, Caplan M, Hsueh W. Endogenous nitric oxide protects against platelet- 24 Caplan MS, Jilling T. New concepts in necrotizing enterocolitis. Curr Opin Pediatr activating factor-induced injury in the rat. Pediatr Res 1993; 34: 222–228. 2001; 13: 111–115. 8 Cintra AE, Martins JL, Patricio FR, Higa EM, Montero EF. Nitric oxide levels in the 25 Neu J. Arginine supplementation for neonatal necrotizing enterocolitis: are we ready? intestines of mice submitted to ischemia and reperfusion: L-arginine effects. Br J Nutr 2007; 97: 814–815. Transplant Proc 2008; 40: 830–835. 26 Batsha ML. Long term treatment of inborn errors of urea synthesis. In: Kleinberger G, 9 Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc Rhoads J et al. Arginine metabolism Ferenci P, Riederer P, Thaler H (eds). Advances in hepatic encephalopathy and urea and nutrition in growth, health and disease. Amino Acids 2009; 37: 153–168. cycle disease. Karger: Basel (Switzerland), 1984, pp 187–195. 10 Batshaw ML, Wachtel RC, Thomas GH, Starrett A, Brusilow SW. Arginine-responsive 27 Urea Cycle Disorders Conference Group. Consensus statement from a conference for asymptomatic hyperammonemia in the premature infant. J Pediatr 1984; 105: 86–91. the management of patients with urea cycle disorders. J Pediatr 2001; 138(1 Suppl): 11 He Q, Kong X, Wu G, Ren P, Tang H, Hao F et al. Metabolomic analysis of the response S1–S5. of growing pigs to dietary L-arginine supplementation. Amino Acids 2009; 37: 28 Fagerberg UL, Loof L, Merzoug RD, Hansson LO, Finkel Y. Faecal calprotectin levels in 199–208. healthy children studied with an improved assay. J Pediatr Gastroenterol Nutr 2003; 12 Wu G, Jaeger LA, Bazer FW, Rhoads JM. Arginine deficiency in preterm infants: 37: 468–472. biochemical mechanisms and nutritional implications. J Nutr Biochem 2004; 15: 29 Campeotto F, Butel MJ, Kalach N, Derrieux S, Aubert-Jacquin C, Barbot L et al. High 442–451. faecal calprotectin concentrations in newborn infants. Arch Dis Child Fetal Neonatal 13 Nankervis CA, Giannone PJ, Reber KM. The neonatal intestinal vasculature: Ed 2004; 89: F353–F355. contributing factors to necrotizing enterocolitis. Semin Perinatol 2008; 32: 83–91. 30 Campeotto F, Kalach N, Lapillonne A, Butel MJ, Dupont C, Kapel N. Time course of 14 Zamora SA, Amin HJ, McMillan DD, Kubes P, Fick GH, Bu¨tzner JD et al. Plasma faecal calprotectin in preterm newborns during the first month of life. Acta Paediatr L-arginine concentrations in premature infants with necrotizing enterocolitis. J Pediatr 2007; 96: 1531–1533. 1997; 131: 226–232. 31 Campeotto F, Baldassarre M, Butel MJ, Viallon V, Nganzali F, Soulaines P et al. Fecal 15 Becker RM, Wu G, Galanko JA, Chen W, Maynor AR, Bose CL et al. Reduced serum calprotectin: cutoff values for identifying intestinal distress in preterm infants. J Pediatr amino acid concentrations in infants with necrotizing enterocolitis. J Pediatr 2000; Gastroenterol Nutr 2009; 48: 507–510. 137: 785–793. 32 Nissen AC, van Gils CE, Menheere PP, Van den Neucker AM, van der Hoeven MA, Forget 16 Richir MC, Siroen MP, van Elburg RM, Fetter WP, Quik F, Nijveldt RJ et al. Low plasma PP. Fecal calprotectin in healthy term and preterm infants. J Pediatr Gastroenterol concentrations of arginine and asymmetric dimethylarginine in premature infants with Nutr 2004; 38: 107–108. necrotizing enterocolitis. Br J Nutr 2007; 97: 906–911. 33 Yang Q, Smith PB, Goldberg RN, Cotten CM. Dynamic change of fecal calprotectin in 17 Neu J. Arginine supplementation and the prevention of necrotizing enterocolitis in very very low birth weight infants during the first month of life. Neonatology 2008; 94: low birth weight infants. J Pediatr 2002; 140: 389–391. 267–271. 18 Amin HJ, Zamora SA, McMillan DD, Fick GH, Butzner JD, Parsons HG et al. Arginine 34 Kapel N, Campeotto F, Kalach N, Baldassarre M, Butel MJ, Dupont C. Faecal supplementation prevents necrotizing enterocolitis in the premature infant. J Pediatr calprotectin in term and preterm neonates. J Pediatr Gastroenterol Nutr 2010; 51: 2002; 140: 425–431. 542–547. 19 Ballard JL, Novak KK, Driver M. A simplified score for assessment of fetal maturation of 35 Laforgia N, Baldassarre ME, Pontrelli G, Indrio F, Altomare MA, Di Bitonto G. newly born infants. J Pediatr 1979; 95(5 Part 1): 769–774. Calprotectin levels in meconium. Acta Paediatr 2003; 92: 463–466.

Journal of Perinatology Journal of Perinatology (2013) 33, 134–140 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Early caffeine therapy and clinical outcomes in extremely preterm infants

RM Patel1,2, T Leong3, DP Carlton1,2 and S Vyas-Read1,2,4 1Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA; 2Children’s Healthcare of Atlanta, Atlanta, GA, USA; 3Departments of Biostatistics and Bioinformatics, Grace Crum Rollins School of Public Health, Emory University, Atlanta, GA, USA and 4The Children’s Center for Developmental Lung Biology, Atlanta, GA, USA

hospital in the first year of life.3 Despite the significant morbidities Objective: To determine if early caffeine (EC) therapy is associated with associated with BPD, few safe and effective therapies are available decreased bronchopulmonary dysplasia (BPD) or death, decreased treatment to prevent the disease.4,5 In addition to BPD, a persistent PDA of patent ductus arteriosus (PDA), or shortened duration of ventilation. is a common problem affecting approximately half of neonates Study Design: In a retrospective cohort of 140 neonates p1250 g at <29 weeks gestation with over two-thirds receiving drug therapy birth, infants receiving EC (initiation <3 days of life) were compared for closure and approximately one-fourth requiring surgical 2 with those receiving late caffeine (LC, initiation X3 days of life) using closure. The presence of a persistent PDA is associated with 6 logistic regression. increased neonatal morbidity such as prolonged ventilation and, although controversial, may also increase the risk of Result: Of infants receiving EC, 25% (21/83) died or developed BPD developing BPD.7,8 compared with 53% (30/57) of infants receiving LC (adjusted odds ratio Given the adverse outcomes associated with BPD and PDA, (aOR) 0.26, 95% confidence interval (CI) 0.09 to 0.70; P<0.01). PDA therapies targeted at reducing or preventing these morbidities are of required treatment in 10% of EC infants versus 36% of LC infants (aOR significant value. In the Caffeine for Apnea of Prematurity (CAP) 0.28, 95%CI 0.10 to 0.73; P ¼ 0.01). Duration of mechanical ventilation trial, caffeine therapy or placebo was initiated during the first 10 was shorter in infants receiving EC (EC, 6 days; LC, 22 days; P<0.01). days of life (DOL) in infants weighing 500 to 1250 g at birth.9 Conclusion: Infants receiving EC therapy had improved neonatal Infants treated with caffeine had a decreased incidence of both BPD outcomes. Further studies are needed to determine if caffeine prophylaxis and PDA when compared with placebo. In addition, caffeine should be recommended for preterm infants. therapy reduced the duration of mechanical ventilation by Journal of Perinatology (2013) 33, 134–140; doi:10.1038/jp.2012.52; approximately 1 week. Importantly, caffeine therapy also resulted published online 26 April 2012 in improved long-term neurodevelopmental outcomes.10 A post-hoc analysis of the CAP trial suggested that the efficacy of caffeine may Keywords: methylxanthine therapy; bronchopulmonary dysplasia; 11 patent ductus arteriosus; neonatal outcomes be dependent on the timing of initiation of study drug. However, the trial only included infants who met inclusion criteria by DOL 10, potentially excluding a significant group of infants who did not Introduction meet clinical criteria for treatment. Despite improvements in survival for very low birth weight infants Early caffeine (EC) therapy may carry additional benefit during over the past two decades, bronchopulmonary dysplasia (BPD) and a critical period of susceptibility to both lung and brain injury in patent ductus arteriosus (PDA) remain common morbidities the first few days of a premature infant’s life. We compared infants affecting this vulnerable population.1 Over 40% of very low birth p1250 g at birth who received caffeine early in their hospital weight infants develop BPD based on recent estimates2 and BPD course with those who were treated later to determine if the timing remains the most common chronic lung disease of infancy. of caffeine therapy would impact common morbidities of prematurity. Neonates with BPD are at high risk of long-term lung disease, Our primary hypothesis was that extremely preterm infants who adverse neurodevelopmental outcomes and readmission to the receive EC therapy (initiation before DOL 3) would have a decreased incidence of BPD or death, when compared with infants who were Correspondence: Dr RM Patel, Division of Neonatology, Emory University School of Medicine, treated with caffeine later in their hospital course (initiation at or Uppergate Dr NE, 3rd floor, Atlanta, GA 30322, USA. after DOL 3). As secondary outcomes, we evaluated if EC initiation E-mail: [email protected] Received 7 February 2012; revised 22 March 2012; accepted 2 April 2012; published online would be associated with a reduction in the treatment of PDA and a 26 April 2012 decreased duration of endotracheal ventilation. Early caffeine therapy and clinical outcomes RM Patel et al 135

Methods diagnosis of NEC made by an attending neonatologist. Surgical Patient population NEC was determined by the need for either exploratory This retrospective cohort study was performed at a single, regional laparotomy or peritoneal drain placement. Postnatal steroid referral level III neonatal intensive care unit (Grady Memorial use was defined as the receipt of either hydrocortisone or Hospital, Atlanta, GA, USA). Infants born between January 2008 and dexamethasone. June 2010 were eligible for inclusion in the study if they met the following three criteria: (1) birth weight (BW) p1250 g (2) BPD estimator treatment with caffeine citrate at any point during hospital course To allow for a validated estimation of the baseline risk of (3) admission to our unit within 24 h after birth. Exclusion criteria moderate-to-severe BPD for patients in the study, a web-based BPD included lack of information regarding timing of caffeine therapy outcome estimator was utilized to estimate the probability of BPD or permanent transfer to another center for continued care. using baseline and early clinical variables (available at https:// An electronic medical record database (NeoData, Isoprime neonatal.rti.org).12 The BPD estimator was formulated using data Corporation, Lisle, IL, USA) was queried to generate a list of all from the National Institute of Child Health and Human patients meeting the inclusion criteria. Further data was obtained Development (NICHD) Neonatal Research Network (NRN) centers, by individual chart review. Appropriate oversight and approval was of which Grady Memorial Hospital is a member. The estimator obtained from the Emory University Institutional Review Board and included the following clinical variables that were predictive of BPD Grady Memorial Hospital Research Oversight Committee. or death: GA, BW, sex, race, ventilator type and fraction of inspired oxygen. First, estimated probabilities of moderate-to-severe BPD, Definitions using variables obtained on both postnatal days 1 and 3 to account Outcomes were compared by the timing of initiation of caffeine for any early changes in postnatal predictor variables, were therapy with ‘early’ defined as initiation before DOL 3 and ‘late’ evaluated for individual patients in the cohort. Next, the mean- defined as initiation at or after DOL 3. We selected DOL 3 as the predicted probability of moderate-to-severe BPD was compared with cutoff to divide the two groups, a priori, based on the median day observed rates of BPD for the entire cohort. Last, the mean- of caffeine initiation in the CAP trial. The day of caffeine initiation predicted probability of BPD was compared between neonates was determined by the DOL at which the patient received the first receiving early and late caffeine (LC) therapy to evaluate for any dose of caffeine therapy, with the day of birth considered DOL 0. potential difference in the baseline risk of BPD. Caffeine therapy was initiated at the discretion of the attending neonatologist. BPD was defined as the requirement of any Statistical analysis supplemental oxygen at a postmenstrual age (PMA) of 36 weeks or PASW 18.0 for Windows (IBM, Armonk, NY, USA) was used for all on the last day of hospitalization for infants discharged before 36 statistical analyses. Median values with interquartile ranges were weeks PMA. In-hospital mortality was calculated for all patients used for continuous variables, unless indicated otherwise. Statistical who died before discharge. Pharmacologic treatment for PDA was significance for unadjusted comparisons was performed using chi- determined by the need for either indomethacin or ibuprofen square or Fisher’s exact tests for categorical variables and Wilcoxon therapy for closure of a PDA after DOL 3. The cutoff using DOL 3 or Student’s t-tests for continuous variables. For outcome analyses, was performed to distinguish between those infants who received logistic regression analysis with adjustment for covariates that are prophylactic indomethacin therapy from those who were treated for predictors of neonatal morbidity and mortality were included in a persistent PDA. To account for infants with early mortality that separate models fit to the primary and secondary outcomes. For the precluded potential treatment of a PDA, neonates who died during outcome of death or BPD, the following covariates were entered the first 7 DOL were not included in the outcome analysis for PDA into the model: BW, GA, sex, twin gestation, race, antenatal steroids requiring treatment. The duration of endotracheal ventilation was (two doses), chorioamnionitis, surfactant therapy and outborn. determined for all infants who were ventilated at the initiation Interaction between covariates was evaluated in the model and did of caffeine therapy and the end of ventilation was defined as no not significantly affect the outcome. The following covariates were endotracheal ventilation for at least 1 day. Gestational age (GA) entered into the model and fit to the outcome of PDA requiring was determined by the best obstetrical estimate using the date of treatment: BW, GA, outborn and surfactant therapy. Our unit has the last menstrual period and/or dating ultrasound. The presence set protocols for the use of prophylactic indomethacin therapy for of intraventricular hemorrhage and periventricular leukomalacia the prevention of intraventricular hemorrhage in infants <1000 g were evaluated by routine ultrasound imaging interpreted by an at birth and prophylactic vitamin A therapy for the prevention attending pediatric radiologist. Retinopathy of prematurity was of BPD in infants <1250 g at birth. As BW was included as a identified by routine screening. Infection was defined as a positive covariate in each of the models, further adjustment for the use blood culture and treatment with antibiotics for at least 7 days. of prophylactic vitamin A and indomethacin therapy was not Medical necrotizing enterocolitis (NEC) was determined by a performed. Covariates with a P-value of <0.10 or which improved

Journal of Perinatology Early caffeine therapy and clinical outcomes RM Patel et al 136 the predictability of the model were included in the final model for Table 1 Patient characteristics the outcome of death or BPD and PDA requiring treatment. Characteristics Early caffeine Late caffeine P-value Statistical significance was defined as a P<0.05. (

Number of patients 83 57 Results Initiation of caffeine therapy (DOL) Median 1 6 <0.01 Over a 30-month period, 176 infants who weighed p1250 g at Interquartile range 0–1 4–15.5 birth were admitted to the Grady Memorial Hospital neonatal intensive care unit and 140 of these infants met the criteria for Duration of caffeine therapy (days) inclusion into the study. Although caffeine treatment was required Median 40 39.5 0.60 for inclusion into this study, we gathered summary data for the Interquartile range 21–58 28–61 infants who were p1250 g at birth and did not meet criteria for Gestational age (weeks) inclusion. Of the 36 infants who were excluded, 14 (39%) died Median 27.3 26.6 0.03 before hospital discharge and never received caffeine therapy. The Interquartile range 25.6–28.7 25.3–27.7 median GA and BW for these infants were 24.5 weeks and 620 g, Birth weight (g) respectively. The remaining 22 infants who were excluded tended to Median 940 910 0.19 be of relatively larger GA and BW (median GA 30.2 weeks and BW Interquartile range 730–1100 715–1035 1045 g). The majority of these infants did not meet inclusion criteria because they never received caffeine therapy, likely because Sex they had little, if any, apnea. The remainder of these 22 infants Male 31 (37%) 27 (47%) 0.24 were excluded because of permanent transfer to another center, Female 52 (63%) 30 (53%) which prevented evaluation of the primary outcome, or because Race they were admitted to our facility after 24 h of life. For the cohort of White 8 (10%) 5 (9%) 0.52 140 infants who met inclusion and exclusion criteria, the median Black 59 (71%) 46 (81%) day of caffeine initiation was DOL 2 (interquartile range 1 to 5 Hispanic 9 (11%) 4 (7%) days) and the mean day of caffeine initiation was DOL 5.3 (s.e.m. Other 7 (8%) 2 (3%) ±0.81). For the entire cohort of 140 infants, the median GA was Twin gestation 12 (15%) 6 (11%) 0.49 27.0 weeks (interquartile range 25.5 to 28.1 weeks) and the median Antenatal steroidsa 39 (47%) 27 (47%) 0.97 BW was 910 g (interquartile range 722 to 1069 g). Chorioamnionitis 12 (15%) 7 (12%) 0.71 Apgar at 1 min (median) 4 4 0.66 Early versus late caffeine groups Apgar at 5 min (median) 7 7 0.86 We divided the cohort into two groups based on timing of caffeine Outbornb 3 (4%) 10 (18%) <0.01 initiation. Of the 83 infants in the EC group, the median day of Abbreviation: DOL, day of life. initiation was DOL 1 and 80% of infants received their initial dose aOnly includes infants who received a complete course of antenatal steroids. of caffeine by DOL 1 (Table 1). For the 57 infants in the LC group, bOnly includes outborn infants who were admitted by 24 h of life. the median day of initiation was DOL 6 with a wide interquartile Data are presented as n (%) unless indicated otherwise. range (4 to 15.5 postnatal days). The median duration of caffeine therapy was similar between the EC and LC groups (EC: 40 days, LC: 39.5 days, P ¼ 0.60). characteristics, both outborn and GA were included as covariates in our statistical models. Patient characteristics Next, we evaluated neonatal morbidities and hospitalization Although infants in the EC group were of slightly older GA (EC: characteristics between those infants who received early and late 27.3 weeks, LC: 26.6 weeks, P ¼ 0.03), there was no significant initiation of caffeine (Table 2). A similar proportion of infants in difference in BW between infants in the EC and LC groups (EC: both groups required mechanical ventilation on DOL 0 and 1, 940 g, LC: 910 g, P ¼ 0.19; Table 1). There were no significant and there was no significant difference in surfactant therapy differences in sex, race, twin gestation, antenatal steroid use, between the groups. There were no significant differences in chorioamnionitis, or 1 and 5 min Apgar scores between groups. the rates of intraventricular hemorrhage, periventricular Infants in the LC group were more likely to be outborn leukomalacia, retinopathy of prematurity or infection between (EC: 3.6%, LC: 17.5%, P<0.01), although this cohort only groups. Additionally, there were no significant differences included outborn infants who were transferred to our center by in the rates of intestinal perforation, NEC or postnatal steroid use 24 h of life. To account for these differences in baseline between the groups.

Journal of Perinatology Early caffeine therapy and clinical outcomes RM Patel et al 137

Neonatal outcomes 36 weeks PMA (Supplementary Figure 1). In-hospital mortality Infants who received EC initiation had a significantly decreased did not differ significantly between groups (EC: 6.0%, LC: 5.3%, incidence of the primary outcome of death or BPD when compared P ¼ 0.64). with those infants with later initiation (EC: 25.3%, LC: 52.6%, Secondary outcomes included the presence of a PDA requiring P<0.01) and this difference remained significant after adjustment treatment and the duration of endotracheal ventilation. Infants for important predictors of BPD (adjusted odds ratio 0.26, 95% receiving EC had a significantly decreased need for pharmacologic confidence interval 0.09 to 0.70; Table 3). This effect was or surgical treatment of a PDA when compared with infants accounted for by the decreased incidence of BPD in those infants receiving later therapy (EC: 10.4%, LC: 36.4%, P ¼ 0.01; Table 3). treated with EC (EC: 23.6%, LC: 50.9%, P ¼ 0.04). In addition, To evaluate the association between total median duration of infants receiving EC had lower levels of respiratory support at endotracheal ventilation and timing of caffeine therapy, we evaluated the duration of endotracheal ventilation in infants who Table 2 Neonatal morbidities and hospital course were ventilated at the initiation of caffeine therapy. The duration of endotracheal ventilation was significantly lower in infants receiving Variables Early caffeine Late caffeine P-value EC compared with LC (EC: 6 days, LC: 22 days, P<0.01). The (stage 1 20 (30%) 21 (41%) 0.20 death (EC: 52%, LC: 94%, P<0.01) while being closely matched in ROP requiring laser surgery 2 (3%) 5 (10%) 0.24 both GA (EC: 25.1 weeks, LC: 25.0 weeks, P ¼ 0.54) and BW (EC: Infection 16 (19%) 17 (30%) 0.15 620 g, LC: 610 g, P ¼ 0.80). The frequency of PDA requiring Intestinal perforation 6 (7%) 3 (5%) 0.74 treatment was significantly lower in infants with a BW between 750 Medical NEC 8 (10%) 10 (18%) 0.15 and 999 g, and the duration of ventilation was significantly lower Surgical NEC 1 (1%) 3 (5%) 0.30 in each BW subgroup. Postnatal steroid usea 11 (13%) 12 (21%) 0.22 Finally, the BPD estimator was used to assess the probability of Abbreviations: DOL, days of life; IVH, intraventricular hemorrhage; NEC, necrotizing developing moderate-to-severe BPD using clinical characteristics enterocolitis; PVL, periventricular leukomalacia; ROP, retinopathy of prematurity. aIncludes the use of both dexamethasone and hydrocortisone. from the first few days of life (postnatal day 1 and 3) to compare Data are presented as n (%). the baseline risk of BPD between groups (Table 5). For the entire

Table 3 Neonatal outcomes by timing of caffeine initiation

Outcomes Early caffeine Late caffeine Odds ratio Adjusted odds ratio P-value (

Primary outcome Death or BPDa 21 (25%) 30 (53%) 0.31 (0.15–0.63) 0.26 (0.09–0.70) <0.01 Death 5 (6%) 3 (5%) 1.15 (0.26–5.03) 1.47 (0.30–7.26) 0.640 BPDb 17 (24%) 27 (51%) 0.30 (0.14–0.64) 0.33 (0.11–0.98) 0.04

Secondary outcomes PDA requiring treatmentc,d 8 (10%) 20 (36%) 0.20 (0.08–0.51) 0.28 (0.10–0.73) 0.01 Pharmacologic only 7 (9%) 15 (27%) Surgical 1 (1%) 5 (9%) Duration of ventilation (median days)e 6 22 <0.01

Abbreviations: BPD, bronchopulmonary dysplasia; CI, conﬁdence interval; DOL, days of life; PDA, patent ductus arteriosus. aOutcome adjusted for gestational age, birthweight, sex, chorioamnionitis, surfactant therapy and antenatal steroid use. bOne infant died after developing BPD. cOutcome adjusted for gestational age, birthweight and outborn. dEight infants were not included in the analysis (four died within 7 days of birth, four lacked details regarding PDA treatment). eFor infants ventilated at initiation of caffeine therapy. Data are presented as n (%) unless indicated otherwise.

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Table 4 Neonatal outcomes by timing of caffeine initiation and by birthweight Table 5 Predicted probability of BPD subgroup Predicted and observed Early caffeine Late caffeine P-value risk of BPD All infants Early caffeine Late caffeine P-valuea (

Subgroup characteristics Probability of BPD on Gestational age, median (weeks) postnatal day 1 31% 29% 32% 0.32 <750 g 25.1 25.0 0.54 (mean)b,c 750–999 g 26.9 26.1 0.16 Probability of BPD on 1000–1250 g 28.7 27.7 <0.01 postnatal day 3 31% 30% 33% 0.43 (mean)b,c Birth weight, median (g) Observed incidence of <750 g 620 610 0.80 BPDd 31% 24% 51% <0.01 750–999 g 860 879 0.58 1000–1250 g 1120 1073 0.06 Abbreviations: BPD, bronchopulmonary dysplasia; DOL, days of life. aComparison of mean probability of BPD between early and late caffeine groups Subgroup outcomes performed using Student’s t-test. b Death or BPD, n (%)a Prediction of BPD by postnatal age uses NIH definition and derived from web-based <750 g 11 (52) 17 (94) <0.01 estimator (available at https://neonatal.rti.org). cOf the 140 infants, 15 were not included. Four infants lacked data regarding fraction of 750–999 g 9 (31) 10 (53) 0.14 inspired oxygen, 11 infants had variables outside of range for web-based calculator (9 1000–1250 g 1 (3) 3 (15) 0.15 with race/ethnicity outside of range and 2 with gestational age outside range). d b For observed incidence, BPD was defined as any oxygen requirement at 36 weeks PDA requiring treatment, n (%) postmenstrual age (study definition). <750 g 4 (22) 6 (35) 0.47 750–999 g 2 (8) 11 (61) <0.001 1000–1250 g 2 (6) 3 (15) 0.35 later therapy. Importantly, this difference remained significant after

c adjusting for baseline differences between groups, including Duration of ventilation, median (days) primary predictors of neonatal morbidity and mortality.13 <750 g 10 29 0.04 Additionally, infants <750 g who are considered to be at the 750–999 g 3 20 <0.01 1000–1250 g 2 7 <0.01 highest risk for BPD or death, demonstrated the strongest association between EC initiation and decreased incidence of Abbreviations: BPD, bronchopulmonary dysplasia; DOL, days of life; PDA, patent ductus BPD or death. We utilized the BPD estimator to further characterize arteriosus. aOne infant died after developing BPD. the baseline risk of BPD between both EC and LC groups. bEight infants were not included in the analysis (four died within 7 days of birth, four The results from the BPD estimator reassured us that infants lacked details regarding PDA treatment). who received EC therapy did not represent a group that had a cFor infants ventilated at initiation of caffeine therapy. lower baseline risk of BPD. Given the limited number of therapies available for targeting cohort, the mean-predicted probability of moderate-to-severe BPD the prevention of BPD, optimizing the use of a therapy that has was similar to the observed incidence of BPD, confirming the utility been shown to be both safe and effective in reducing BPD may be of the BPD estimator in this population of infants. Next, we an ideal strategy to decrease the burden of neonatal respiratory assessed the probability of moderate-to-severe BPD in the EC and morbidity. Caffeine is a potential drug of choice for the prevention LC groups using baseline variables and predictor variables obtained of BPD in very low birth weight infants.4 In addition, caffeine is on both postnatal days 1 and 3. The mean probability of one of the most commonly used medications in the neonatal developing moderate–to-severe BPD was similar between infants intensive care unit,14 and it remains a highly cost-effective treated with EC and LC. In contrast, the observed incidence of BPD therapy.15 Mechanistically, EC initiation may decrease pulmonary was significantly different between groups, suggesting that EC may morbidity by improving pulmonary function and enhancing potentially modify the risk of moderate-to-severe BPD. central respiratory drive. Caffeine therapy has been shown to improve lung function by improving minute ventilation,16 pulmonary mechanics17,18 and respiratory muscle contractility.19 Discussion Additional potential benefits of methylxanthines include the In this study, early initiation of caffeine therapy before DOL 3 in protection of lung tissue against damage from injury.20,21 Our infants p1250 g at birth was associated with decreased neonatal findings suggest that early initiation during a period of critical morbidity. Infants who received EC had approximately one-half the susceptibility to injury may be necessary to confer the maximal incidence of BPD or death when compared with infants undergoing benefit of caffeine therapy.

Journal of Perinatology Early caffeine therapy and clinical outcomes RM Patel et al 139

Interestingly, we found that EC initiation was associated with Although the retrospective study design prohibits the establishment a decreased incidence of PDA requiring treatment. Potential of causality and differences in patient characteristics may have mechanisms of action for this effect include diuresis and altered influenced the timing of caffeine initiation, we attempted to fluid balance,22 increased cardiac output and blood pressure23 account for these differences in baseline characteristics through or an improvement in overall pulmonary mechanics. Although both adjustments in our statistical models and the use of subgroup caffeine may also affect several signaling molecules involved analyses. However, we acknowledge our inability to control for all in ductal constriction, investigations on this effect are still factors that are reflective of early severity of illness or that inconclusive.24–26 Possibly, improved respiratory morbidity in potentially influenced the clinical outcomes. Our findings of the neonate reduces the likelihood of a PDA requiring decreased BPD in infants receiving EC, although indicative treatment, although the management of pharmacological and of a substantial benefit for at-risk preterm infants, remain surgical ductal closure remains a controversial area of hypothesis-generating and necessitate additional investigation practice.27 including validation in a large, multicenter cohort of The physiologic effects of caffeine may increase the success patients. of early initial continuous positive airway pressure therapy or In conclusion, our results demonstrate that EC initiation is facilitate weaning from the ventilator and result in a reduction of associated with decreased BPD in extremely preterm infants. Given endotracheal ventilation and protection against associated lung the limited number of safe and well-studied therapies that are injury. These potential benefits of caffeine therapy are supported by effective in decreasing the burden of BPD,4,5 caffeine remains a findings in our study in which EC initiation was associated with an potential first-line therapy of choice for the prevention of BPD approximately 2 week decrease in the duration of endotracheal in preterm infants. Optimizing the timing of caffeine therapy by ventilation. Our findings are consistent with results from the CAP initiating earlier treatment or by instituting universal prophylaxis trial in which caffeine treatment resulted in approximately 1 week may carry additional benefits over its conventional use as a less of positive pressure ventilation.9 In addition, a recent Cochrane treatment for apnea of prematurity or for facilitation of extubation. analysis concluded that methylxanthine prophylaxis increases the Our data suggest that the treatment effect of EC use may be chance of successful extubation within 1 week of treatment.28 substantial compared with later initiation. However, randomized In our study, neonates successfully discontinued the use of controlled trials of caffeine prophylaxis to prevent neonatal endotracheal ventilation within 4 to 5 days following initiation of morbidities, such as BPD and PDA, are necessary to conclusively caffeine therapy, regardless of whether they received early or late support the routine use of caffeine as a preventative therapy and caffeine initiation. to ensure the safety of early initiation of caffeine in extremely Although our study did not identify any potential adverse effects preterm infants. associated with the early initiation of caffeine therapy, the small sample size limited detection of clinically significant differences. Prior studies have raised concerns regarding the vasoconstrictive Conflict of interest 29 effects of caffeine therapy. Potentially, these vasoconstrictive The authors declare no conflict of interest. effects may be amplified by concomitant administration of prophylactic indomethacin therapy, which also has vasoconstrictive properties.30 However, a large, multicenter randomized controlled Acknowledgments trial did not identify any safety concerns related to the use of We thank Becky Kinkead for her critical review of this manuscript. caffeine citrate.9,10 Limitations of this study include its design as a single-center, retrospective cohort study with a predominately African-American References population. In addition, we were unable to ascertain the indication 1 Fanaroff AA, Stoll BJ, Wright LL, Carlo WA, Ehrenkranz RA, Stark AR et al. Trends in for caffeine therapy in each patient and no institutional protocol neonatal morbidity and mortality for very low birthweight infants. Am J Obstet Gynecol directed caffeine use. Although we did not evaluate long-term 2007; 196(2): 147 e141-e148. 2 Stoll BJ, Hansen NI, Bell EF, Shankaran S, Laptook AR, Walsh MC et al. Neonatal effects in our study, caffeine treatment has been shown to outcomes of extremely preterm infants from the NICHD Neonatal Research Network. reduce the incidence of long-term neurologic impairment and Pediatrics 2010; 126(3): 443–456. cerebral palsy.10 The role of caffeine in protection against 3 Baraldi E, Filippone M. Chronic lung disease after premature birth. N Engl J Med neurodevelopmental impairment may be explained, in part, by 2007; 357(19): 1946–1955. its effect on improving respiratory morbidity. However, a recent 4 Schmidt B, Roberts R, Millar D, Kirpalani H. Evidence-based neonatal drug therapy for prevention of bronchopulmonary dysplasia in very-low-birth-weight infants. investigation demonstrating improved white matter microstructural Neonatology 2008; 93(4): 284–287. development in caffeine-treated infants suggests that other 5 Laughon MM, Smith PB, Bose C. Prevention of bronchopulmonary dysplasia. Semin 31 unidentified mechanisms of action are likely to play a role. Fetal Neonatal Med 2009; 14(6): 374–382.

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6 Cotton RB, Stahlman MT, Bender HW, Graham TP, Catterton WZ, Kovar I. 18 Yoder B, Thomson M, Coalson J. Lung function in immature baboons with respiratory Randomized trial of early closure of symptomatic patent ductus arteriosus in small distress syndrome receiving early caffeine therapy: a pilot study. Acta Paediatr 2005; preterm infants. J Pediatr 1978; 93(4): 647–651. 94(1): 92–98. 7 Brooks JM, Travadi JN, Patole SK, Doherty DA, Simmer K. Is surgical ligation of patent 19 Supinski GS, Deal Jr EC, Kelsen SG. The effects of caffeine and theophylline on ductus arteriosus necessary? The Western Australian experience of conservative diaphragm contractility. Am Rev Respir Dis 1984; 130(3): 429–433. management. Arch Dis Child Fetal Neonatal Ed 2005; 90(3): F235–F239. 20 Li J, Li G, Hu JL, Fu XH, Zeng YJ, Zhou YG et al. Chronic or high dose acute caffeine 8 Marshall DD, Kotelchuck M, Young TE, Bose CL, Kruyer L, O’Shea TM. Risk factors for treatment protects mice against oleic acid-induced acute lung injury via an adenosine chronic lung disease in the surfactant era: a North Carolina population-based study of A2A receptor-independent mechanism. Eur J Pharmacol 2011; 654(3): 295–303. very low birth weight infants. North Carolina Neonatologists Association. Pediatrics 21 Oliveira-Junior IS, Pinheiro BV, Silva ID, Salomao R, Zollner RL, Beppu OS. 1999; 104(6): 1345–1350. Pentoxifylline decreases tumor necrosis factor and interleukin-1 during high tidal 9 Schmidt B, Roberts RS, Davis P, Doyle LW, Barrington KJ, Ohlsson A et al. Caffeine volume. Braz J Med Biol Res 2003; 36(10): 1349–1357. therapy for apnea of prematurity. N Engl J Med 2006; 354(20): 2112–2121. 22 Gillot I, Gouyon JB, Guignard JP. Renal effects of caffeine in preterm infants. Biol 10 Schmidt B, Roberts RS, Davis P, Doyle LW, Barrington KJ, Ohlsson A et al. Long-term Neonate 1990; 58(3): 133–136. effects of caffeine therapy for apnea of prematurity. N Engl J Med 2007; 357(19): 23 Soloveychik V, Bin-Nun A, Ionchev A, Sriram S, Meadow W. Acute hemodynamic 1893–1902. effects of caffeine administration in premature infants. J Perinatol 2009; 29(3): 11 Davis PG, Schmidt B, Roberts RS, Doyle LW, Asztalos E, Haslam R et al. Caffeine for 205–208. Apnea of Prematurity trial: benefits may vary in subgroups. J Pediatr 2010; 156(3): 24 Blaustein MP, Golovina VA, Song H, Choate J, Lencesova L, Robinson SW et al. 382–387. Organization of Ca2+ stores in vascular smooth muscle: functional implications. 12 Laughon MM, Langer JC, Bose CL, Smith PB, Ambalavanan N, Kennedy KA et al. Novartis Found Symp 2002; 246: 125–137; discussion 137-141, 221-227. Prediction of bronchopulmonary dysplasia by postnatal age in extremely premature 25 Whorton AR, Collawn JB, Montgomery ME, Young SL, Kent RS. Arachidonic acid infants. Am J Respir Crit Care Med 2011; 183(12): 1715–1722. metabolism in cultured aortic endothelial cells. Effect of cAMP and 3-isobutyl-1- 13 Tyson JE, Parikh NA, Langer J, Green C, Higgins RD. Intensive care for extreme methylxanthine. Biochem Pharmacol 1985; 34(1): 119–123. prematurity–moving beyond gestational age. N Engl J Med 2008; 358(16): 1672–1681. 26 Clyman RI, Roman C. The effects of caffeine on the preterm sheep ductus arteriosus. 14 Clark RH, Bloom BT, Spitzer AR, Gerstmann DR. Reported medication use in the Pediatr Res 2007; 62(2): 167–169. neonatal intensive care unit: data from a large national data set. Pediatrics 2006; 27 Hamrick SE, Hansmann G. Patent ductus arteriosus of the preterm infant. Pediatrics 117(6): 1979–1987. 2010; 125(5): 1020–1030. 15 Dukhovny D, Lorch SA, Schmidt B, Doyle LW, Kok JH, Roberts RS et al. 28 Henderson-Smart DJ, Davis PG. Prophylactic methylxanthines for endotracheal Economic evaluation of caffeine for apnea of prematurity. Pediatrics 2011; 127(1): extubation in preterm infants. Cochrane Database Syst Rev 2010; (12): CD000139. e146–e155. 29 Hoecker C, Nelle M, Poeschl J, Beedgen B, Linderkamp O. Caffeine impairs cerebral and 16 Aranda JV, Turmen T, Davis J, Trippenbach T, Grondin D, Zinman R et al. Effect intestinal blood flow velocity in preterm infants. Pediatrics 2002; 109(5): 784–787. of caffeine on control of breathing in infantile apnea. J Pediatr 1983; 103(6): 30 Van Bel F, Van de Bor M, Stijnen T, Baan J, Ruys JH. Cerebral blood flow velocity 975–978. changes in preterm infants after a single dose of indomethacin: duration of its effect. 17 Davis JM, Bhutani VK, Stefano JL, Fox WW, Spitzer AR. Changes in pulmonary Pediatrics 1989; 84(5): 802–807. mechanics following caffeine administration in infants with bronchopulmonary 31 Doyle LW, Cheong J, Hunt RW, Lee KJ, Thompson DK, Davis PG et al. Caffeine and dysplasia. Pediatr Pulmonol 1989; 6(1): 49–52. brain development in very preterm infants. Ann Neurol 2010; 68(5): 734–742.

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Journal of Perinatology Journal of Perinatology (2013) 33, 98–102 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Prenatal diagnosis of criss-cross heart: sonographical and pathological features of ﬁve cases

SLi1, G Luo2,3, ER Norwitz4, C Wang5, S Ouyang6, Y Yao1, H Wen1, C Chen1,QFu1, X Xia1,JBi1 and J Zhu7 1Department of Ultrasound, Shenzhen Maternity and Child Healthcare Hospital, Affiliated to Southern Medical University, Shenzhen, China; 2Department of Obstetrics and Gynecology, Danbury Hospital, Danbury, CT, USA; 3Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA; 4Department of Obstetrics and Gynecology, Tufts University School of Medicine, Boston, MA, USA; 5Department of Obstetrics and Gynecology, Shenzhen Maternity and Child Healthcare Hospital, Affiliated to Southern Medical University, Shenzhen, China; 6Department of Laboratory Medicine, Shenzhen Maternity and Child Healthcare Hospital, Affiliated to Southern Medical University, Shenzhen, China and 7Department of National Center for Birth Defects Monitoring, West China Second University Hosiptal, Chengdu, China

Introduction Objective: To describe the sonographical and pathological features of fetal Criss-cross heart (CCH), first described by Lev and Rowlatt in 1961, criss-cross heart (CCH). is a rare complex congenital cardiac anomaly characterized by Study Design: All cases of fetal CCH diagnosed by fetal echocardiogram distorted atrioventricular (AV) connections and crossed ventricular from May 2003–May 2011 were identified at a single referral center using inflow streams.1 In such cases, the spatial arrangement of the an established perinatal database. Demographic and genetic information, ventricles is altered early in embryonic development owing to either sonographical images and autopsy reports were reviewed. Sonographical clockwise rotation or counter-clockwise rotation of the ventricles and pathological features are described. along the cardiac axis (Figure 1). The reported incidence of CCH is Result: Five cases of fetal CCH were identified, all of which were B8 per 1 000 000 live births and it accounts for <0.1% of all 2 confirmed by autopsy. Characteristic sonographical findings include: (1) congenital heart defects. the inability to obtain four-chamber view at standard transverse plane Accurate prenatal diagnosis is important for prenatal through the fetal chest; (2) appreciation of the misaligned spatial atrial– counseling, and postnatal surgical and cardiorespiratory ventricle connection with the interventricular septum in a ‘spiraling’ management as early palliative surgery appears to have a favorable 3 orientation; (3) orientation of the two ventricular inlets in a superior– impact on functional cardiac status in adulthood. However, inferior and crossing position; and (4) a four-chamber-like view seen in the distorted cardiac anatomy together with a high prevalence the sagittal plane of the fetal chest. Doppler ultrasound demonstrates the of associated cardiac defects makes prenatal diagnosis very ‘criss-cross’ arrangement of the inflow tracts into the two ventricles challenging. To date, only four cases of prenatally diagnosed CCH 4,5 simultaneously in the transverse plane of the fetal chest. have been reported in the English literature. Here, we report five cases diagnosed prenatally at our institution from May 2003 Conclusion: CCH is a rare developmental disorder that can be accurately through May 2011. The sonographical and pathological features diagnosed prenatally. Early diagnosis will allow for more targeted are discussed in detail. counseling and early intervention. Journal of Perinatology (2013) 33, 98–102; doi:10.1038/jp.2012.56; published online 3 May 2012 Methods Keywords: congenital cardiac defects; criss-cross heart; fetal echocar- The Prenatal Diagnosis Center at Shenzhen Maternity and Child diography; prenatal diagnosis; ultrasonography Healthcare Hospital in Shenzhen, China, was established as a national prenatal diagnosis training center in 2004, and serves as a referral hospital for patients from throughout China. Correspondence: Dr S Li, Department of Ultrasound, Shenzhen Maternity and Child A comprehensive perinatal database was established in which Healthcare Hospital, Affiliated to Southern Medical University, Shenzhen 518028, China. E-mail: [email protected] or Dr C Wang, Department of Obstetrics and Gynecology, prenatal and postnatal ultrasound images of all patients are stored Shenzhen Maternity and Child Healthcare Hospital, Affiliated to Southern Medical University, along with their demographic and clinical information, including Shenzhen 518028, China. pregnancy outcome, results of genetic analysis and autopsy reports E-mail: [email protected] Received 30 November 2011; revised 19 March 2012; accepted 2 April 2012; published online in cases of pregnancy termination. Where possible, data regarding 3 May 2012 the long-term outcome of infants and children born with Prenatal diagnosis of criss-cross heart SLiet al 99

Figure 1 Abnormal ventricular rotation associated with criss-cross heart malformation. At some point in embryonic development after formation of the ventricular loop and interventricular septum, the relationship between the ventricles and atria becomes distorted owing either to: (i) clockwise rotation of the ventricular mass along its long axis in normal positioned ventricles (a), or (ii) counter-clockwise rotation of the ventricular mass along its long axis in isolated inverse ventricles (c). This rotation results in a change in the ventricular spatial relationship, with the interventricular septum orientated in the horizontal plane. The two most common types of CCH malformations are shown (b, d). LA, left atrium; LV, left ventricle; MLV, morphological left ventricle; MRV, morphological right ventricle; RA, right atrium; RV, right ventricle. congenital anomalies are included. For the purposes of this coronal view of the trachea and branches, and a coronal view of study, the database was mined to identify consecutive cases the descending aorta at the junction of the ductus and arch. In all of CCH malformations. This study was approved by the cases, pulsed and color Doppler flow imaging was also performed Institutional Review Board of the Shenzhen Maternity and to confirm patency of the ventricular inflow tracts, outflow tracts Child Healthcare Hospital, which is an affiliation of the and arches, and to assess the flow patterns in the pulmonary veins, Southern Medical University, China. umbilical artery, umbilical vein and ductus venosus. In all cases, All prenatal ultrasound examinations were performed on Acuson the diagnosis was confirmed by postnatal echocardiography for Sequoia machines (Siemens Medical Solutions, Mountain View, CA, liveborn infants or autopsy for terminated cases. All perinatal USA) using 4- or 6-mHz curvilinear or 8-MHz phased array ultrasound and fetal echocardiography examinations were transducers. Fetal echocardiography was performed in all cases performed by physicians specialized in prenatal diagnosis with up where fetal anomalies were detected. Fetal echocardiography to 10 years of experience. includes a high abdominal transverse view, a four-chamber cardiac view (4CV), left and right ventricular outflow tract views and a detailed cardiac assessment, including demonstration of the atrial Results and visceral situs, systemic and pulmonary venous connections, From May 2003 to May 2011, a total of five cases of fetal CCH were symmetric 4CV, patency and symmetry of the AV valves, both identified. Two were referred to our center with suspected fetal outflow tracts and great arteries, and the ductal and aortic arches, cardiac anomalies; the remaining three were detected at our center. including their positions relative to the trachea. If an aortic arch The average maternal age was 29.0±8.6 years (range, 22–43 anomaly is suspected, additional views are obtained, including a years). The average gestational age at diagnosis was 25.0±3.6

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Figure 2 Representative prenatal echocardiographic findings. While the typical four-chamber view of the heart was not able to be imaged in transverse plane of the fetal chest, the left ventricular inflow tract (LVIT) (a) and right ventricular inflow tract (RVIT) (b) could be displayed by tilting the transducer and sweeping from the upper abdomen to the heart, respectively. In this case, the LVIT lined up from left-posterior to right-anterior position and was inferior to the RVIT (a), while the latter aligned from right-posterior to left-anterior position (b). The spatial relationship of the two ventricular inflow tracts therefore ended up in a supero-inferior and crossing position. Because of their orientation, color Doppler flow imaging could display the two crossing blood streams of the LVIT and RVIT simultaneously in the transverse plane of the fetal chest giving the pathognemonic criss-cross appearance (c). As the ventricles were in a supero-inferior position and the interventricular septum in a horizontal position, the four-chamber view could be obtained only with a sagittal section of the fetal chest (d). DAO, descending aorta; L, left; LA, left atrium; LV, left ventricle; R, right; RA, right atrium; RV, right ventricle; SP, spine; VSD, ventricular septal defect.

Table 1 The pathological features, karyotype result and outcome of fetal CCH

Case MA (years) GA Atrial Venous–atrial Atrialventricular Ventricular–aortic connection Ventricular Karyotype Outcome (weeks) situs connection connection arrangement

1 23 26 Solitus Normal Concordant Complete TGA Supero-inferior Normal TOP 2 22 21 Solitus Normal Concordant PTA Supero-inferior Normal TOP 3 43 22 Solitus Normal Concordant DORV + PA Supero-inferior Normal TOP 4 31 30 Solitus Normal DILV Complete TGA + PS Supero-inferior Normal TOP 5 26 26 Solitus Normal Concordant DORV + PS Supero-inferior Not checked TOP

Total 29.0±8.6 25.0±3.6 All ﬁve solitus Normal ¼ 5 Concordant ¼ 4; Complete TGA ¼ 1; PTA ¼ 1; Supero-inferior ¼ 5 Normal ¼ 4; one TOP ¼ 5 DILV ¼ 1 DORV+ PA ¼ 1; complete case not checked TGA+PA ¼ 1; DORV +PS ¼ 1

Abbreviations: DILV, double inlet left ventricle; DORV, double outlet right ventricle; GA, Gestational age; MA, Maternal age; PA, pulmonary atresia; PS, pulmonary stenosis; PTA, persistent truncus arteriosus; TGA, transposition of great arteries; TOP, termination of pregnancy. Data are given as mean±s.e.m. weeks (range, 21–30 weeks). All of the patients were nulliparous left and right atria, the transducer needed to be tilted and swept and none reported any signiﬁcant medical conditions or family carefully from the upper abdomen in a cephalad direction. Careful histories. evaluation revealed the spatial relationship between the cardiac In all ﬁve cases, the 4CV of the heart in the transverse plane chambers. The left atrium was located to the left, posterior and through the fetal chest appeared abnormal (Figure 2a and b). To inferior, and the left ventricle to the right, posterior and inferior of obtain adequate views of the AV valves, left and right ventricles, and their expected positions. Similarly, the right atrium was located to

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Figure 3 Pathological findings associated with criss-cross heart malformation of case 1. At autopsy, a ventral view of the cardiac anatomy (a) showed that the atria were in situs solitus, the right atrial appendage (RAA) was above the left atrial appendage (LAA), the right ventricle (RV) was superior to the left ventricle (LV), the right ventricular apex pointed to the left, the LV was inferior to the RV, the left ventricular apex pointed to the right side and the aorta was in front of the pulmonary artery with a dextrorotated aortic arch. Highlighting was used to graphically illustrate the left ventricular inflow tract (in yellow (LVIT)) and right ventricular inflow track (in blue (RVIT)) (b). In this case, the LVIT was inferior to the RVIT, and the criss-crossing of the two ventricular inflow tracks can be easily visualized. After sectioning the RV along the right side of the anterior interventricular and atrioventricular grooves (c), we observed that the aorta arose from the RV, the RVIT aligned from right-posterior to left-anterior position, the interventricular septum (IVS) was in horizontal position and a large ventricular septal defect (VSD) was evident. After sectioning the LV along the left side of the anterior interventricular and atrioventricular grooves, and stretching the IVS in an anterior and superior direction(d), we observed that the main pulmonary artery (MPA) arose from the LV, the pulmonary valve (PV) was significantly stenosed, the LVIT was aligned from the left-posterior to right-anterior position and a large VSD was confirmed. ATV, anterior cusp of tricuspid valve; LIA, left innominate artery; LIV, left innominate vein; MV, mitral valve; RCA, right carotid artery; RIV, right innominate vein; RSA, right subclavian artery; RVAW, right ventricular anterior wall; SVC, superior vena cava; T, trachea. the right, posterior and superior position, and the right ventricular inlets was in superior–inferior and crossing ventricle to the left, anterior and superior of their expected position and the interventricular septum (IVS) is in spiraling positions. The mitral valve was located posteriorly and inferiorly, orientation, which is why the 4CV in the transverse plane and the tricuspid valve anteriorly and superiorly of their of the fetal chest was abnormal in all cases. However, a 4C-like expected positions (Figure 2). Instead of running in parallel in the view could be displayed in the sagittal plane through the fetal chest standard 4CV, the left ventricle inflow tract (LVIT) and right (Figure 2d). ventricle inflow tract (RVIT) are seen to be crossing (Figure 2a All five cases had other associated heart defects, including and c). Color Doppler studies clearly demonstrated the ‘criss-cross’ ventricular septal defect (n ¼ 5 (VSD)), complete transposition of appearance of the two AV inlet tracts (Figure 2c). The orientation the great arteries (n ¼ 2 (TGA)), double outlet right ventricle of blood flow in the LVIT is aligned from left-posterior to (n ¼ 1 (DORV)), truncus arteriosus (n ¼ 1), pulmonary stenosis right-anterior, while the orientation of the blood flow in the (n ¼ 3) and/or pulmonary atresia (n ¼ 1) (Table 1). Additional RVIT is aligned from right-posterior to left-anterior. The RVIT is non-cardiac anomalies included two cases of single umbilical superior to the LVIT. Thus, the spatial relationship of the two artery and one case of right microtia.

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In all five cases, the couple chose to proceed with elective The prognosis for the fetus depends on the associated cardiac as termination of pregnancy after extensive counseling. Autopsy was well as non-cardiac anomalies. Isolated CCH with adequate four- performed on all five cases, and the diagnosis of CCH was chamber development may require only palliative surgery after confirmed in each instance. The characteristic pathological birth. There are little data on long outcome. As previously reported, features of CCH are shown in Figure 3. In four cases, most cases of CCH are associated with other complex cardiac amniocentesis revealed a normal karyotype; one patient declined defects, and most cases diagnosed prenatally ended in elective karyotype analysis (Table 1). termination of pregnancy (Table 1). In the current case series, we described the characteristic sonographical and pathological features of CCH, and demonstrate Discussion that CCH can be accurately diagnosed prenatally. Although rare, Although initially described in 1961,1 the term ‘criss-cross heart’ accurate prenatal diagnosis will allow for more targeted counseling (CCH) first appeared in the literature in two manuscripts published and will give couples options that may not be available after in 1974.6,7 Almost all published cases of CCH to date are from delivery. diagnoses made in neonates and children ranging in age from 1 month to 25 years.8,9 Here, we report five cases diagnosed prenatally and confirmed at autopsy. Conflict of interest CCH is caused by abnormal rotation of the ventricular mass The authors declare no conflict of interest. along its long axis during embryonic development, while the ventricular loop and IVS are formed normally and the base of heart 6 remains fixed (Figure 1). Anderson et al proposed that the two Acknowledgments most common types of CCH are generated by clockwise rotation of the ventricular mass along its long axis with complete TGA (Figure This study was funded by the research of prenatal imaging screening and new 1a and b) and counter-clockwise rotation of the ventricular mass diagnostic technologies for major fetal structural and anomalies, and supported along its long axis with corrected TGA (Figure 1c and d), by National Key Technologies R&D Program (No. 2006BAI05A04). respectively. This abnormal rotation produces the spiraling IVS and malalignment of the atrial and ventricular septum, while the latter results in a large VSD below the AV valves. The orientation of References the ventricular rotation often correlates with the AV connections. 1 Lev M, Rowlatt UF. The pathologic anatomy of mixed levocardia. A review of thirteen If the ventricles rotated clockwise, the AV connections tended to cases of atrial or ventricular inversion with or without corrected transposition. Am J concordance and complete TGA and DORV may be combined. On Cardiol 1961; 8: 216–263. 2 Fyler DC. Trends. In: Fyler DC, (ed). Nada’s Pediatric Cardiology. Philadelphia PA: the other hand, if the ventricles rotated counter-clockwise, the AV Hanley& Belfus, 1992, p. 273–280. connections tended to discordance and corrected TGA and DORV 3 Snider AR, Enderlein MA, Teitel DF, Hirji M, Heymann MA. Isolated ventricular may be combined. inversion:two-dimensional echocardiographic findings and a review of the literature. Prenatal diagnosis of CCH is critical to provide accurate Ped Cardiol 1984; 5: 27–33. counseling to the couple and to plan for early postnatal surgery, if 4 Ngeh N, Api O, Iasci A, Ho SY, Carvalho JS. Criss-cross heart: report of three cases with double-inlet ventricles diagnosed in utero. Ultrasound Obstet Gynecol 2008; 31: indicated. However, prenatal diagnosis is difficult, especially in the 461–465. presence of other complex cardiac anomalies. By sweeping and 5 Sklansky MS, Lucas VW, Kashani IA, Rothman A. Atrioventricular situs concordance with tilting the ultrasound transducer from the upper abdomen in a atrioventricular alignment discordance: fetal and neonatal echocardiograhic findings. cephalad direction, one can demonstrate the key sonographical Am J Cardiol 1995; 76: 202–204. features of CCH. These include: (1) an inability to obtain a 4CV at 6 Anderson RH, Shinebourne EA, Gerlis LM. Criss-cross atrioventricular relationships the standard transverse plane through the fetal chest; (2) an producing paradoxical atrioventricular concordance or discordance. Their significance to nomenclature of congenital heart disease. Circulation 1974; 50: appreciation of the spatial atrial–ventricle connection with the IVS 176–180. in a spiraling orientation; (3) orientation of the two ventricular 7 Ando M, Takao A, Cho E, Vehara K, Nihmural I. Criss-cross heart by abnormal rotation inlets in a superior–inferior and crossing position; and (4) a 4C- of bulboventricular loop: diagnostic considerations for complex cardiac anomaly. Proc like view seen in the sagittal plane of the fetal chest. Color Doppler Ped Circ Soc 1974; 4: 82–83. ultrasound provides critical information by visualizing the ‘criss- 8 Hoffman P, Szymanski P, Lubiszewska B, Rozanski J, Lipczynska M, Klisiewicz A. Crisscross hearts in adults: echocardiographic evaluation and natural history. J Am Soc cross’ arrangement of the inflow tracts into the two ventricles Echocardiogr 2009; 22: 134–140. simultaneously in the transverse plane of the fetal chest 9 Yang YL, Wang XF, Cheng TO, Xie MX, Lu Q, Lu XF et al. Echocardiographic (Figure 2c). characteristics of the criss-cross heart. Int J Cardiol 2010; 140: 133–137.

Journal of Perinatology Journal of Perinatology (2013) 33, 103–106 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Signiﬁcance of neonatal body indices in identifying fetal macrosomia

Y Song1,2, S Zhang1 and W Song1 1Department of Gynecology and Obstetrics, Shengjing Hospital of China Medical University, Liaoning, PRC and 2Department of Gynecology and Obstetrics, Benxi Central Hospital of China Medical University, Liaoning, PRC

as birth weight X4000 g, there are no satisfactory clinical Objective: To explore the significance of neonatal body indices in indicators that allow one to identify those infants who may have identifying macrosomic infants, we compared the neonatal birth weight, higher risk of adverse outcomes. Because of an abnormal body length, head circumference, chest circumference, neonatal body metabolic environment, macrosomic infants born to diabetic indices, Quetelet index (QI), Kaup Index (KI), head circumference to chest mothers may develop asymmetrically as reflected by increased circumference ratio (HC/CC) and maternal fasting blood glucose (BG) for ratios of body weight (BW), length (BL), and chest circumference. both diabetic and healthy mothers. Detection of such infants using fetal ultrasound indices may Study Design: This is a retrospective study of 177 macrosomic neonates, identify ‘at risk’ infants for possible referral to a tertiary center for 100 having been born to normal mothers and 77 to diabetic mothers. care. Previous studies have used the Quetelet index (QI ¼ BW 3 Multiple regression analyses were done between neonatal body indices and (kg)/BL (cm) 10 ), which is the weight divided by BL in 2 4 maternal fasting BG. centimeters, and the Kaup Index (KI ¼ BW (kg)/BL (cm) 10 ), which is the weight divided by BL per square centimeter. QI and KI Result: Fetal QI and KI indices of macrosomic infants of diabetic use mean relative BW, which can be used to reflect body density mothers were higher compared with those born to healthy mothers, and evaluate individual girth, width and body organization density. whereas the HC/CC was the reverse. The multiple regression equation used The QI and KI have been used to measure adult adiposity and to compare neonatal physical development indices to maternal fasting BG adolescent nutritional status.4,5 Although QI, KI and other indices was BG ¼ 6.959 þ 0.031QI(4.482 HC/CC). QI and HC/CC had linear are used to evaluate fetal growth at various gestational ages in relationships to maternal fasting BG (P<0.05). As birth weight had no China, its validity in macrosomic infants remains unproven. direct correlation with maternal fasting BG, it was not introduced into the Hence, our objective was to evaluate several fetal body indices to regression equation. possibly identify those infants who may be at higher risk. We Conclusion: Higher QI and KI along with lower HC/CC may be a retrospectively analyzed QI, KI and HC/CC (head circumference to predictor of macrosomia due to maternal diabetes when compared with chest circumference ratio) in macrosomic infants delivered by both birth weight alone. healthy and diabetic mothers. Journal of Perinatology (2013) 33, 103–106; doi:10.1038/jp.2012.58; published online 31 May 2012 Methods Keywords: gestational diabetes mellitus; macrosomia; neonatal body indices All infants were delivered at ShengJing Hospital from May 2009 to July 2010. Complete medical records were available for 177 macrosomic infants, 100 of which were born to normal mothers Introduction and 77 to mothers with gestational diabetes mellitus (GDM). BW, A recent study suggested an increase in the prevalence of BL, head circumference and chest circumference were measured macrosomia.1 Macrosomic infants born to diabetic mothers have a for all infants and recorded by nurses in the newborn nursery. higher likelihood of perinatal complications when compared with For macrosomic infants delivered by healthy mothers, their those born to normal mothers.2,3 Although macrosomia is defined mothers’ 50 g oral glucose challenge test (GCT), oral glucose tolerance test and blood glucose (BG) level were normal. In Correspondence: Professor W Song, Department of Gynecology and Obstetrics, Shengjing addition, their mothers had no complications during the Hospital of China Medical University, No. 36 San Hao Street, Liaoning 110004, PRC. gestational period. In the macrosomic infants delivered by diabetic E-mail: [email protected] or [email protected] Received 26 April 2011; revised 13 February 2012; accepted 12 April 2012; published online mothers, their mothers had no previous history of diabetes or other 31 May 2012 complications and were diagnosed with GDM during pregnancy. Significance of neonatal body indices in fetal macrosomia Y Song et al 104

Table 1 The neonatal body indices and maternal fasting glucose of macrosomic infants delivered by healthy and diabetic mothers

Group Case BW (kg) QI KI HC/CC BG (mmol l1)

Healthy 100 4.269±0.209 80.764±4.874 15.308±1.427 1.007±0.038 4.456±0.604 Diabetic 77 4.340±0.261 82.704±5.782 15.787±1.560 0.993±0.043 5.712±1.725 P–value 0.118 0.002 0.028 0.030 0.000

Abbreviations: BG, maternal fasting blood glucose; HC/CC, head circumference/chest circumference; KI, Kaup Index; QI, Quetelet index. Data are listed as mean±s.d.

The GDM screening test was done between 24 and 28 weeks of mothers. Measurement data are demonstrated as the pregnancy by administering a 50 g oral GCT. If one’s GCT result mean±standard deviation. was abnormal (X7.8 mmol l1), further testing with a 75 g oral We took the neonatal physical development indices of the two glucose tolerance test over 2 h was performed. A diagnosis of GDM groups as the baseline measurement for overall and pair-wise is defined as a maternal fasting plasma glucose X5.1 mmol l1, comparisons and came to the conclusion that for both groups, 1 h result X10.0 mmol l1 and/or 2 h result X8.5 mmol l1.6 when birth weight X4000 g, there was a statistical difference Other inclusion criteria were single gestation, full term in QI, KI and HC/CC between macrosomic infants delivered (gestational age X37 weeks), and no pregnancy-related by healthy and diabetic mothers. Macrosomic infants delivered complications (for example, pregnancy-induced hypertension). by diabetic women had significantly higher QI and KI than The following maternal variables were also recorded: mode of that of macrosomic infants delivered by healthy women delivery, maternal fasting plasma glucose, 50 g GCT result, oral (P<0.01, P<0.05), while lower HC/CC was seen in macrosomic glucose tolerance test result and calculated QI, KI and HC/CC. infants delivered by diabetic mothers (P<0.05). The fasting Neonatal variables recorded were: hypoglycemia, respiratory BG was significantly different between the two groups distress, the 1-min Apgar score, shoulder dystocia and other (P<0.01). complications among infants admitted to the Neonatal Intensive Care Unit (NICU). The correlation between QI, KI, HC/CC and maternal fasting BG Shoulder dystocia is diagnosed when delivery professionals are Neonatal birth weight, QI, KI and HC/CC for all 177 macrosomic required to use delivery techniques other than conventional infants were designated as independent variables, whereas traction and episiotomy to deliver the shoulders. Hypoglycemia was maternal fasting BG was set as the dependent variable in our diagnosed if the neonatal heel stick BG level was <2.2 mmol l1 at multiple regression analysis. Our study showed that the the time the newborn was taken to the nursery. Respiratory distress correlation coefficient of QI and HC/CC demonstrated stronger was defined as a neonate with tachypnea and cyanosis requiring correlation than neonatal birth weight when compared with supplemental oxygen or assisted ventilation. Neonates were maternal fasting BG. In multiple regression equation, using admitted to the NICU secondary to birth trauma and/or various introduced gradually–eliminate method, only QI and HC/CC immediate adverse outcomes. had been introduced into regression model. Its coefficient of Data were analyzed using SPSS13.0 (SPSS, Chicago, IL, USA) determination, R, is 0.299, and adjustment for the determination software. If the measurement data could not satisfy normal coefficient is 0.082. The multiple regression equation of body distribution and comparison of the mean homogeneity of variance, indices to maternal fasting BG is BG ¼ 6.959 þ 0.031QI we used Kruskal–Wallis and Mann–Whitney U-tests. The rest of (4.482 HC/CC). QI and HC/CC have linear relationships with mean measurement data comparison used one-way analysis of maternal fasting BG (P<0.05). Birth weight has no direct variance and least significant difference test. Proportions were correlation with maternal fasting BG and is not introduced into analyzed with the w2 test where appropriate. The analysis of the regression equations. correlation between the neonatal physical indicators and maternal Table 2 displays maternal Cesarean section rates and BG was performed using the multiple regression analysis method. neonatal outcomes in macrosomic infants delivered by healthy and diabetic mothers. When comparing these two kinds of macrosomia, significant differences were found in the incidence of hypoglycemia (50.6%), shoulder dystocia (6.5%) and admission Results to NICU (11.7%). No significant differences were found between The QI, KI and HC/CC in macrosomic infants delivered by the two groups in the incidence of respiratory distress and 1-min healthy and diabetic mothers Apgar score <7. Furthermore, diabetic mothers with macrosomia Table 1 summarizes the comparisons of body indices between had a significantly higher tendency to require delivery by Cesarean macrosomic infants delivered by both healthy and diabetic section (67.5%).

Journal of Perinatology Signiﬁcance of neonatal body indices in fetal macrosomia Y Song et al 105

Table 2 Maternal cesarean section rate and neonatal outcomes between length ratio, a decrease in head circumference to shoulder macrosomic infants delivered by healthy and diabetic mothers circumference ratio and a decrease in HC/CC. Healthy Diabetic P-value (n ¼ 100) No. (%) (n ¼ 77) No. (%) Maternal fasting BG and macrosomia For pregnant women, fasting BG in early pregnancy acts as an Cesarean section 38 (38.0) 52 (67.5) <0.001 important predictive factor in macrosomia.14,15 Higher fasting first Hypoglycemia 4 (4.0) 39 (50.6) <0.001 trimester BG levels significantly increase the risk for adverse Shoulder dystocia 1 (1.0) 7 (9.1) 0.028 pregnancy outcomes, including macrosomia.16 Women with Admission to NICU 2 (2.0) 9 (11.7) 0.021 impaired fasting glucose were more likely to have an large for Respiratory distress 4 (4.0) 8 (10.4) 0.094 14 Apgar score <7 at 1-min 1 (1.0) 4 (5.2) 0.225 gestational age infant than those without glucose impairment. Simultaneously, the incidence of neonatal complications (such as Abbreviation: NICU, neonatal intensive care unit. fetal distress, malformations and Erb’s palsy) was also increased.17 Discussion Macrosomic infants delivered by mothers with GDM are The significance of QI, KI and HC/CC and their clinical value in primarily due to maternal hyperglycemia and fetal identifying macrosomic infants delivered by diabetic mothers hyperinsulinism. Maternal hyperglycemia may cause Newborns with birth weight X4000 g and without perinatal morphological changes in the placenta in affected women. Studies 7 neonatal complications are called ‘normal’ macrosomic infants. revealed increasing angiogenesis and chorionic villous branching As macrosomic infants delivered by gestational diabetic pregnant seen in the human placenta of maternal diabetic pregnancies,18 8 women are at higher risk of complications, we need more which provided a greater surface for maternal–fetal blood 9 information in order to distinguish whether a macrosomic infant exchange and facilitated the passage of glucose to the fetus, thereby is delivered by a gestational diabetic or a healthy pregnant woman. explaining fetal macrosomia. After passing through the feto- Aside from BW, further body indices are required. placental barrier, the mother’s glucose induces the release of The lack of a universally accepted definition of macrosomia insulin from the fetal pancreas, leading to fetal hyperinsulinism.19 10 remains a problem. In China, macrosomia is defined as birth Studies also examined the implications of multiple growth factors weight X4000 g. However, some cases of macrosomia may be and their receptors in GDM and macrosomia,18–20 which may have affected by the interactions of genetics and other factors and could an influence on the transport of glucose across the placenta develop ultimately into healthy infants with symmetrical and lead to the eventual delivery of a macrosomic infant. proportions, although their birth length is greater than average As discussed above, there is significant correlation between a and they weigh more than 4000 g. Our study attempted to provide a higher maternal fasting BG level and the delivery of a macrosomic possible foundation for identifying macrosomic infants delivered by infants, which also leads to the increasing incidence of perinatal diabetic mothers by introducing some rarely used body indices, and other complications in these infants. such as QI, KI and HC/CC, in order to better reflect neonatal physical development characteristics. The correlation of QI and HC/CC to maternal fasting BG is 11 In the 1980s, the neonatal physical development research significantly higher than that of neonatal birth weight to collaboration group of 15 cities in China did some tentative studies maternal fasting BG and partially established a normal range and evaluation criteria for The correlation coefficients of QI and HC/CC were higher in maternal neonatal physical development indicators.12 QI, KI and HC/CC were fasting BG than in neonatal birth weight, suggesting that QI and HC/ among them, as they are able to reflect the newborns’ development in CC may better reflect fetal intrauterine energy metabolism. This leads detail. However, there is still no report on how the above body indices us to conclude that for macrosomic infants whose mothers’ BG level apply in macrosomia. The aim of our study is to investigate whether is unclear during delivery, QI, HC/CC and KI should be paid great there is any difference in these neonatal body indices between attention. For macrosomic infants with higher QI and KI but lower macrosomic infants delivered by healthy and diabetic mothers. HC/CC, a close eye should be kept on future development. For future We concluded that in the two groups of macrosomic infants studies, via a prospective calculation and statistic analysis, we will try with birth weight X4000 g, QI, KI and HC/CC were of great to provide a definitive value for the above indices to evaluate statistical significance. In addition, macrosomic infants delivered macrosomic infants delivered by diabetic mothers. by diabetic mothers showed a disproportionate growth pattern, with overgrowth of insulin-sensitive tissues, such as adipose and muscle, The adverse prognostic incidence of macrosomic infants whereas the growth of insulin-insensitive organs, such as the brain delivered by diabetic mothers is significantly higher than that of and kidneys, did not increase conspicuously.13 This character macrosomic infants delivered by healthy mothers represented the tendency of subcutaneous fat deposition toward Our study came to the conclusion that the incidence of shoulders and limbs and led to an increase in birth weight to birth hypoglycemia, NICU admission and shoulder dystocia in

Journal of Perinatology Significance of neonatal body indices in fetal macrosomia Y Song et al 106 macrosomic infants delivered by diabetic mothers was significantly 2 Shirima CP, Kinabo JL. Nutritional status and birth outcomes of adolescent pregnant higher than those delivered by healthy mothers. Because of poor girls in Morogoro, Coast and Dar es Salaam regions, Tanzania. Nutrition 2005; 21: glycemic control, the diabetic pregnant women were subjected to 32–38. hyperglycemia, which in turn led to fetal hyperinsulinemia. After 3 Lin F, Wu Y. The clinical analysis on non-diabetic macrosomia. Yi Xue Yan Jiu Sheng delivery, excessive neonatal insulin resulted in neonatal Xue Bao 2008; 21: 60–62. 4 Khosla T, Lowe CR. Indices of obesity derived from body weight and height. Br J Prev hypoglycemia. In addition, fetal hyperinsulinemia was also Soc Med 1967; 21: 122–128. associated with hyperplasia of adipose tissue in the shoulders, 5 Billewicz WZ, Kemsley WF, Thomson AM. Indices of adiposity. Br J Prev Soc Med 1962; leading to shoulder dystocia. Birth trauma and immediate neonatal 16: 183–188. outcomes mandated a higher incidence of NICU admissions, which 6 International Association of Diabetes and Pregnancy Study Groups Consensus Panel. 21,22 was consistent with several previous studies. Regarding the International association of diabetes and pregnancy study groups recommendations on increased incidence of respiratory distress and 1-min Apgar score the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care 2010; <7, one possible explanation could be the high cesarean section 33: 676–682. rate in these infants. Through retrospective analysis, we found that 7 Kale SD, Kulkarni SR, Lubree HG, Meenakumari K, Deshpande VU, Rege SS et al. the cesarean section rate for macrosomic infants delivered by Characteristics of gestational diabetic mothers and their babies in an Indian diabetes diabetic mothers is up to 67.5% (52/77). With the help of strict clinic. J Assoc Physicians India 2005; 53: 857–863. 8 Agarwal MM, Dhatt GS, Punnose J, Koster G. Gestational diabetes: dilemma caused by glycemic control and antenatal care, our hospital has seen a recent multiple international diagnostic criteria. Diabet Med 2005; 22: 1731–1736. downward trend in this ratio. 9 Sacks DA, Chen W. Estimating fetal weight in the management of macrosomia. Obstet As our study is retrospective, the relative information from Gynecol Surv 2000; 55: 229–239. mothers could only be collected from the case records. As BG might 10 Cunningham FG (2000) Williams Obstetrics. Beijing Kexue Press: Beijing, 2000, only reflect one aspect of maternal and fetal energy metabolism, our pp 568–572. future research will be prospective in nature and will focus on the 11 Zhang LH, Zhang BL. Physical development analysis of fetus whose gestational age relationship between the above body indices, along with more indices between 28–44 weeks in fifteen cities of China. Xin Sheng Er Za Zhi 1989; 4: 97. 12 Liu XH, Zhang BL. Analysis on neonatal body indices of newborns with different and their effect on maternal metabolic indicators such as HbA1C. gestational age in China. Xin Sheng Er Za Zh 1998; 13: 105–107. Our research demonstrated that the two types of macrosomic 13 Hermann GM, Dallas LM, Haskell SE, Roghair RD. Neonatal macrosomia is an infants, both with birth weight X4000 g, had no difference in BL, independent risk factor for adult metabolic syndrome. Neonatology 2010; 98: 238–244. HC and CC; however, QI, KI and HC/CC were of great statistical 14 Legardeur H, Girard G, Mandelbrot L. Screening of gestational diabetes mellitus: a new significance. Meanwhile, the correlation between QI, HC/CC and consensus? Gynecol Obstet Fertil 2011; 39: 174–179. maternal fasting BG was significantly higher than that between birth 15 Riskin-Mashiah S, Damti A, Younes G, Auslender R. First trimester fasting weight and maternal fasting BG. QI, KI and HC/CC may be better hyperglycemia as a predictor for the development of gestational diabetes mellitus. ways to distinguish asymmetrical, large babies with macrosomia- Eur J Obstet Gynecol Reprod Biol 2010; 152: 163–167. related syndrome. Our next analysis of this group will provide the 16 Black MH, Sacks DA, Xiang AH, Lawrence JM. Clinical outcomes of pregnancies complicated by mild gestational diabetes mellitus differ by combinations of abnormal cutoff points of QI, KI and HC/CC in order to enhance the oral glucose tolerance test values. Diabetes Care 2010; 33: 2524–2530. identification of macrosomic infants delivered by diabetic mothers. 17 Fadl HE, Ostlund IK, Magnuson AF, Hanson US. Maternal and neonatal outcomes and time trends of gestational diabetes mellitus in Sweden from 1991 to 2003. Diabet Med 2010; 27: 436–441. Conflict of interest 18 Leach L. Placental vascular dysfunction in diabetic pregnancies: intimations of fetal The authors declare no conflict of interest. cardiovascular disease? Microcirculation 2011; 18: 263–269. 19 Grissa O, Yessoufou A, Mrisak I, Hichami A, Amoussou-Guenou D, Grissa A. Growth factor concentrations and their placental mRNA expression are modulated in Acknowledgments gestational diabetes mellitus: possible interactions with macrosomia. BMC Pregnancy Childbirth 2010; 9: 10:7. This work was supported by 2010 Educational key laboratory Project in Liaoning 20 Forbes K, Westwood M. Maternal growth factor regulation of human placental province (NO. LS2010164). development and fetal growth. J Endocrino 2010; 207: 1–16. 21 Gyurkovits Z, Ka´llo´ K, Bakki J, Katona M, Bito´ T, Pa´lAet al. Neonatal outcome of macrosomic infants: an analysis of a two-year period. Eur J Obstet Gynecol Reprod References Biol 2011; 159: 289–292. 1 Al-Mendalawi MD. Fetal macrosomia greater than or equal to 4000 grams. Comparing 22 Das S, Irigoyen M, Patterson MB, Salvador A, Schutzman DL. Neonatal outcomes of maternal and neonatal outcomes in diabetic and non-diabetic women. Saudi Med J macrosomic births in diabetic and non-diabetic women. Arch Dis Child Fetal Neonatal 2009; 30: 583. Ed 2009; 94: 419–422.

Journal of Perinatology Journal of Perinatology (2013) 33, 147–153 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Neonatal intensive care unit: predictive models for length of stay

GJ Bender1,3, D Koestler2,3, H Ombao2,3, M McCourt1,3, B Alskinis1,3, LP Rubin1,3,4 and JF Padbury1,3 1DepartmentofPediatrics,Women&Infants’HospitalofRhodeIsland,Providence,RI,USA;2Biostatistics Section, Department of Community Health at Brown University, Providence, RI, USA and 3Warren Alpert Medical School at Brown University, Providence, RI, USA

recognized interest in predicting morbidity and quality of life Objective: Hospital length of stay (LOS) is important to administrators and measures,4 mortality remains the most common outcome for these families of neonates admitted to the neonatal intensive care unit (NICU). scoring systems. Nonetheless, they have driven a wide range of A prediction model for NICU LOS was developed using predictors birth applications, including quality assurance programs,5 triaging weight, gestational age and two severity of illness tools, the score for patients to appropriate services,6 cost effectiveness analyses7,8 and neonatal acute physiology, perinatal extension (SNAPPE) and the morbidity for benchmarking ICU performance.9 Scoring systems have been assessment index for newborns (MAIN). used in clinical decision making,10 stratification for research 11 12–14 Study Design: Consecutive admissions (n ¼ 293) to a New England trials and pediatric trauma outcomes. Length of hospital regional level III NICU were retrospectively collected. Multiple predictive stay (LOS) is often reported as an ancillary or intermediate models were compared for complexity and goodness-of-fit, coefficient of outcome, but has merit in its own right. 15–16 determination (R2) and predictive error. The optimal model was validated Among scores developed specifically for neonatal medicine, prospectively with consecutive admissions (n ¼ 615). Observed and two that have demonstrated sound prediction of morbidity and expected LOS was compared. death are the morbidity assessment index for newborns (MAIN) score,17 and the score for neonatal acute physiology, perinatal Result: The MAIN models had best Akaike’s information criterion, extension (SNAPPE).18 SNAPPE and MAIN were derived from highest R2 (0.786) and lowest predictive error. The best SNAPPE model different populations. The MAIN score was designed to compare underestimated LOS, with substantial variability, yet was fairly well outcomes among high-risk maternal populations or obstetric calibrated by birthweight category. LOS was longer in the prospective healthcare delivery systems.19 It was developed as a discriminative cohort than the retrospective cohort, without differences in birth weight, index for neonates more than 28 weeks gestation, born 1995 gestational age, MAIN or SNAPPE. to 1996 with minimal to moderate neonatal morbidities. Conclusion: LOS prediction is improved by accounting for severity of In contrast, the score for neonatal acute physiology was a illness in the first week of life, beyond factors known at birth. Prospective physiologic organ system-based illness severity score developed validation of both MAIN and SNAPPE models is warranted. to predict mortality in the neonatal intensive care unit (NICU).20 Journal of Perinatology (2013) 33, 147–153; doi:10.1038/jp.2012.62; SNAPPE parsed the original 26 variables into the six most published online 7 June 2012 relevant, augmented with three other variables: birth weight, small for gestational age status and 5-min Apgar scores.21 Keywords: neonatal intensive care units; length of stay; severity of illness index; benchmarking; projections and predictions; score for SNAPPE scoring requires less extensive record keeping than the neonatal acute physiology MAIN score. The goal of the present study is to use these illness severity and morbidity tools to develop a single LOS prediction model valid Introduction across the full spectrum of gestational ages using first week of life A variety of generic clinical scoring systems have been developed to data from which MAIN and SNAPPE were validated. The relative measure severity of illness and to predict mortality.1–3 Despite a contributions from birth weight, gestational age and each severity of illness score are unknown. Accurately modeling individual Correspondence: Dr GJ Bender, Department of Pediatrics, Women & Infants’ Hospital of Rhode Island, 101 Dudley Street, Providence, RI 02905-2499, USA. patient LOS would help manage family expectations, as well as E-mail: [email protected] contribute vitally to NICU resource allocation. Team assignment 4Current address: Department of Pediatrics (Neonatology), University of South Florida cohorts, staff scheduling, resource costs and revenues correlate College of Medicine, Tampa, FL, USA. Received 19 October 2011; revised 20 April 2012; accepted 30 April 2012; published online with NICU census. Census, as the accumulation of current 7 June 2012 neonates, is affected by how long each neonate stays in the NICU. NICU predictive length of stay GJ Bender et al 148

Understanding variation in individual LOS may enable NICU Statistical methods census projections, with resultant organizational and financial One-way frequencies were used to summarize the distribution of benefits from effective resource management. MAIN score items. Accelerated failure time parametric survival models22 were used to assess the relationship between LOS and birth weight, gestational age and their second-order terms, as well Methods as the MAIN and SNAPPE morbidity scores. To distinguish the The training set consisted of 293 consecutive NICU admissions, superior scoring system, separate regressions were considered ranging in morbidity from minimal to severe. These were involving MAIN and SNAPPE. SNAPPE scores were modeled both as ascertained using retrospective data collection from chart quantitative and categorical factors (p8, 9 to 12, X13). Neonates abstractions from all newborns born between August and discharged before day 3 of life had a presumed SNAPPE of zero. October 1999 and admitted to our NICU. In order to test the validity To avoid estimation bias, analysis was repeated with a regression imputation approach predicting SNAPPE on day 3 of life for these of the model developed a prospective study was undertaken. 23 The prospectively collected validation data set included 615 patients. Regression models were used to identify the most consecutive admissions, as well as 61 existing NICU patients, relevant variables and their contribution to LOS, based on a from April to September 2002. retrospective data set. Parametric nested model development is described in more detail in Supplementary Appendix 3. Generalized 2 Eligibility coefficients of determination (R ), the Akaike’s information Every viable newborn admitted to the nicu at Women and Infants criterion (AIC) and K-fold cross-validation were used for model 2 Hospital within 24 h of birth was recruited. Newborns were comparison. R estimates how much of outcome variability is excluded if they (1) died before NICU admission; (2) were admitted accounted for by the model. AIC penalizes models with excess 24 for preterminal comfort care (defined as neither intubation nor logistic regression variables that fail to improve prediction error. cardio-respiratory resuscitation); (3) had a major congenital K-fold cross-validation partitions the data to compare each model’s anomaly. inherent predictive ability. Models were examined for subpopulations of very brief hospital Scoring and data collection stays, of birth weight categories and of disposition destination. With IRB approval, data were manually extracted for MAIN and The effect of missing data was explored by repeating analyses on all SNAPPE scores for the training data set, as was SNAPPE for the admissions, by imputing missing data, and by excluding patients validation data set. We collected the 47 items representing 24 for whom complete data were not available. Imputing day 3 neonatal attributes identified as most relevant in the MAIN SNAPPE scores for patients discharged before day 3 was done by validation data set19 over the first day and first 7 days of life. The regression on the population with complete data. LOS by weight SNAPPE score was also assessed for all neonates, for the first and categories were compared with one-tailed student’s t-test. Low and third 24-h periods of life. Examples of our MAIN and SNAPPE data very low birthweight (VLBW) groups were compared with the collection sheets are available on request. Missing data for severity normal birth weight group with marginal generalized R2. of illness scores were reconciled according to the original MAIN Regardless of which model demonstrated best predictive power, and SNAPPE protocols. For example, if a child did not have a blood external validation was planned for the optimal SNAPPE model. An gas on day 3, then they received a ‘0’ for that SNAPPE element. For independent data set was available that had SNAPPE but no MAIN transported infants, the SNAPPE on day 1 of life was calculated data elements. At a minimum, the potential for census prediction for the actual day of life one, even if the child was in a normal using individual LOS could be established. Calibration by patient nursery or in an outlying hospital. disposition and birth weight category was done with observed The gestational age was taken as actual completed gestational minus expected LOS (OMELOS), with expected being the predicted weeks. An infant who was 32 6/7 was recorded as 32 weeks. An median LOS using the best SNAPPE model. Observed and predicted infant who was 33 0/7 was recorded as 33 weeks. Fractional days median LOS was plotted on a logarithmic (base 10) scale for were discarded due to uncertainty of the best obstetric estimate. the overall validation population. Patients who died, were retro- Pediatric assessment of gestational age was used if obstetric transferred to a level II facility, or were transferred out to a level III estimate was not available. Weight was taken from documentation facility were identified and included. in the operating room, delivery room or NICU, whichever was Census projections were calculated from the validation data set. recorded first. Newborns dying during the first 24-h scoring period Observed census on a given day was the accumulation of all were scored, but were analyzed separately. LOS reflects total neonates whose admit date was in the past and discharge date primary hospitalization following their admission to a NICU, in the future. Expected census on a given day includes those incrementing at midnight. Time spent in other facilities before neonates who would still be in the NICU by their predicted discharge home was included in LOS. individual LOS. Observed and expected census (mean±s.d.) was

Journal of Perinatology NICU predictive length of stay GJ Bender et al 149 compared using student’s t-test, and graphed to identify focal died (23.8±8.5 vs 2.5±1.4 days). The mortality rate jumped to trends and outliers. Pearson’s correlation was used to quantify the 45% among neonates with SNAPPE >70 on day 1 of life, consistent association between observed and expected daily census, and was with Richardson’s original population observations.21 Correlation followed by hierarchal linear regression to identify variation due to analysis shows significant interrelationship between MAIN scores daily admissions. Seasonality was examined by controlling for on days 1 and 7, SNAPPE scores on day 1 and 3, birth weight, census date. gestational age and LOS (Supplementary Appendix 2). Regression models (Supplementary Appendix 3) developed using data from 278 admissions found a log-logistic distribution fit best Results for SNAPPE as a quantitative variable, and Weibull fit best for LOS, birth weight, gestational age, discharge type, SNAPPE scores MAIN. Table 1 shows model constituent variables and predictive and MAIN scores for the training (3 months, 1999) and validation ability. Superior models have high R2, low AIC and low prediction (6 months, 2002) populations are shown in Supplementary error by cross validation. Models have incrementally greater Appendix 1. Illness severity score distributions are shown in explanatory power by adding a weight-by-age interaction term, Figure 1. The distribution of day 1 SNAPPE score demonstrated low MAIN or SNAPPE scores, and a SNAPPE day 1-by-day 3 interaction acuity in the majority of neonates, with increasing physiologic term. MAIN model 6a explained the highest proportion of the stability by day 3. MAIN scores increased from day 1 to day 7 as variance in LOS (R2 0.786, AIC 556, cross validation 0.524), as expected for a cumulative scoring system. Infants discharged with compared with the best SNAPPE model 4a (R2 of 0.709, AIC 623, home health services had longer LOS. Average LOS was higher in cross validation 0.575). Analysis of birth weight subgroups the validation population (P<0.04), primarily due to longer upper identified the same pattern, with generalized R2 on MAIN and quartile LOS (28 vs 19 days). Associated factors include VLBW SNAPPE models for normal birth weight (>2500 g, n ¼ 126) of (P<0.05, see Figure 2) and older postnatal age among those who 0.563 and 0.271, for low birthweight (1500 to 2500 g, n ¼ 102) of

a 80% 70% Derivation, Day 1 60% Validation, Day 1 50% Derivation, Day 3 Validation, Day 3 40%

Frequency 30%

20%

10%

0% 4 12202836 44 52 60 68 76 84 92 SNAPPE Score

b 25%

20% Day 1 Week 1 15%

10% Frequency

0% 0 500 1000 1500 2000 2500 3000 MAIN Score Figure 1 Distributions for severity of illness scores: (a) Neonatal acute physiology, perinatal extension (SNAPPE) scores on day 1 and 3 show increasing proportion of stable neonates in both derivation and validation populations. (b) Cumulative morbidity assessment index for newborns (MAIN) scores increase over ﬁrst week.

Journal of Perinatology NICU predictive length of stay GJ Bender et al 150

160

140

120 Derivation Population 100 Validation Population

60 Length of Stay 40

>4000 400-599 600-799 800-999 1000-12491250-14991500-17491750-1999 2000-24992500-2999 3000-34993500-3999 Weight Category Figure 2 Length of stay (LOS) by birthweight category. The difference between derivation and validation populations was not statistically signiﬁcant.

Table 1 Model constituent variables and predictive ability On average, the best SNAPPE model underestimated LOS ± Coefﬁcient variables Model (OMELOS 3.3 41 days, n ¼ 675), even after removing the outlier populations that died or were transferred (2.6±14, 1 2 3 4a4b5a5b6a6b7a7b n ¼ 580). This may reﬂect higher observed LOS in the validation

BW ||||||||||| population, particularly among extremely low birthweight (born GA ||||||||||| <750 g birth weight) neonates. The wide OMELOS variability BW GA |||||among neonates who died (88±195 days, n ¼ 15), reflects BW BW ||| || their exclusion in development of the original prediction models. SNAPPE DOL1 |||| SNAPPE DOL3 |||| There is substantial variability in OMELOS for patients who SNAPPE || were transferred to other level III units (78±106 days, n ¼ 19), DOL1 DOL3 were retro-transferred (9±23 day, n ¼ 61) as well. Fair model MAIN 1 |||| calibration is demonstrated in Figure 3, examining OMELOS by MAIN 7 |||| MAIN 1 MAIN 7 || birth weight subgroup. A log-linear relationship between observed and predicted LOS is shown in Figure 4, by type of hospital Predictive ability disposition. AIC 663 652 642 623 639 632 650 556 558 560 563 Calculated census fluctuations are shown in Figure 5. Observed 2 R 0.66 0.67 0.68 0.71 0.79 census (65.6±5.37) was lower than expected census (67.1±4.60, Cross validation 0.58 0.52 P<0.001) primarily due to discrepancies on days in September. Abbreviations: AIC, Akaike’s information criterion; BW, birthweight; GA, gestational age; Census predictions using individual LOS predictions were MAIN, morbidity assessment index for newborns; SNAPPE, score for neonatal acute physiology, perinatal extension. significantly correlated with observed census (r ¼ 0.25, P<0.01). The correlation between observed and expected census increases (r ¼ 0.46, P<0.001) when controlling for date, suggesting a seasonal effect not accounted for in the prediction. Hierarchal 0.641 and 0.612 and for VLBW (<1500 g, n ¼ 50) of 0.679 linear regression showed that daily admissions accounted for and 0.608, respectively. To account for short stays in the training additional 10% of observed census variation over and above the data set, the analyses were repeated either imputing or setting impact of expected census (DR2 ¼ 0.10, F(1, 180) ¼ 21.03, SNAPPE day of life 3 to 0 for the 50 patients discharged before that P<0.001). day. Neither the variable estimates, model AIC, R2 or prediction error changed notably. External validation was feasible for the SNAPPE models. Discussion OMELOS was calculated for each individual in the validation NICU resource allocation, staffing, costs and revenues all pivot population based on SNAPPE model 4a. around one measure: census. Variability in census requires

Journal of Perinatology NICU predictive length of stay GJ Bender et al 151

150

100

0 0 500 1000 1500 2000 2500 3000 3500 4000 -50

-100 Length of Stay (days)

Observed minus Expected -150

-200 Birthweight Category (>grams) Figure 3 Calibration of neonatal acute physiology, perinatal extension (SNAPPE) model by birthweight category, observed minus expected length of stay (OMELOS). The model overestimated length of stay (LOS) in the smallest neonates, but underestimated LOS for those neonates with birth weight between 1000 and 1750 g.

7 Neonatal deaths been developed in a population of larger neonates (<1% VLBW), Neonates retro-transfered and SNAPPE has demonstrated its best relationship to LOS among Other neonatal transfers 17,18 6 Surviving neonates not transferred populations of smaller neonates (>45% VLBW). We found that models including MAIN were statistically superior to those with 5 SNAPPE overall, as well as within our normal, low birth weight and VLBW populations. This relative performance may result from 4 information details (incorporating 47 vs 9 perinatal factors) or time span (covering 7 days vs 3 days), or both. The density of 3 information detail has practical implications. The superior model was chosen with Aikake’s Information Criterion, which adjusts for log(observed LOS) 2 the number of predictors, but not for the complexity inherent to each predictor. SNAPPE elements may be captured automatically 1 from some electronic medical record systems; the clinical utility of MAIN may be limited unless informatics strategies are developed to 0 acquire the data. 123456 Severity of illness scoring in the first week of life improves LOS log(predicted median LOS) prediction beyond gestational age and birth weight alone, from Figure 4 Observed vs predicted length of stay (LOS) by hospital disposition, 65.5 to 70.9% (using SNAPPE) to 78.6% (using MAIN) of the logarithm10 (days), validation population. Notable outliers include neonates who variance in hospital LOS for this population. Individual LOS died or were transferred to level III facilities, but level II retro-transfers were prediction is less certain, notably shorter than expected among similar to survivors who were not transferred. neonates who died and longer than expected among neonates transferred to another level III facility. Although these sickest neonates represented <5% of the overall population, they as well as persistent attention by administrators, as inefficient resource those excluded for congenital anomalies detract from the strength allocation incurs costs. Although stochastic fluctuations in birth of single-model LOS prediction. Some LOS variance among the rates greatly impact census, other factors also contribute to census large proportion of healthier neonates (week 1 discharges, Figure 4 variability. Once admitted to the NICU, a neonate contributes to lower left corner) is an artifact of extra day accrual at midnight. census for the duration of his stay. Quantifying variation in A neonate born just before midnight has a measured LOS one individual LOS with moderate accuracy would thereby facilitate greater than an equivalent neonate born at 1:00 hours. Rounding NICU resource allocation. It would also help families directly by of hospital days or gestational weeks also create noise in LOS reducing the uncertainty in the response to their most consistent measurement. Avoiding fractions of days to align with hospital question ‘when will he be able to go home?’. accounting systems affects observed LOS, with progressively less We sought a single model to predict LOS across our entire impact on longer stays. Rounding down gestational age implies population as well as possible in the first week of life. MAIN had that up to 6 days in utero has no physiologic impact on severity of

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50 Admissions 40 Observed Census 30

Number of Patients Expected Census 20

Date Figure 5 Fluctuation in observed daily neonatal intensive care unit (NICU) census compared with predicted NICU census, based on accumulating admissions and their predicted lengths of stay (LOS).

illness, which for the youngest neonates is a source of variance in week 1 data, wherein SNAPPE and MAIN were validated. The expected LOS. predictions would likely be enhanced by integrating morbidities Other sources of bias were sought. Seasonal variations cannot be from later in the hospital stay, or possibly by using the early data to excluded given 3-and 6-month data collection periods. Discharge profile risk for these intermediate clinical outcomes. These LOS practice patterns may not align among neonatal attending prediction models should be generalized with caution until tested physicians even with a single institution. Tracking LOS at other in other settings. Severity of illness measures partially compensate facilities enabled correlation of complete hospitalization to for case mix, but differing populations and treatment practices may physiologic stability (retro-transfer squares in Figure 4), reducing significantly affect LOS. measurement bias. Inclusion of pre-existing neonates reduces Census projections built upon individual SNAPPE LOS sampling bias. Exclusion of neonates with congenital anomalies predictions correlated well with observed census. Given markedly may limit application of a single model. Mortality rates may have better variance explained using MAIN LOS predictions, census introduced bias, since non-survival truncates observed LOS, but projections using these would be even more accurate. Census they were similar in our overall (2.1% vs 2.2%) as well as in the was progressively overestimated over the study period, even though extremely low birthweight (23.1% vs 18.8%) population, consistent the individual SNAPPE model underestimated LOS on average. with published neonatal mortality trends in the 1995 to 2002 The offset in September suggests discharge practice variation epoch.25 We did note a shift towards later postnatal age at death, between providers. Our data confirm the correlation between also consistent with the literature.26,27 There were no other daily admissions and census fluctuation. Other variables likely identifiable populations or clinical practice differences in the in affect census trends, including seasonality of admissions and these epochs. Repeating these analyses with a more recent data set, changing discharge practices when approaching bed capacity. one that includes the MAIN variables, may give insight to the Biasing early and late census populations, either by excluding source or persistence of these differences. short stay neonates or overrepresenting sicker neonates, was Prolonged NICU stays are associated with intermediate clinical minimized by tracking neonates for many weeks before and outcomes and social factors that affect LOS. Bannwart28 showed after the study. Modeling NICU census with inclusive LOS prediction models using the entire hospital stay were superior admission and discharge functions may contribute as much to to those using just the first 3 days (R2 0.81 vs 0.60). Cotton29 census projections as individual LOS based on physiologic identified specific morbidities (necrotizing enterocolitis requiring abnormalities. Timely update of individual morbidities, mortality surgery, chronic lung disease at 36 weeks, late-onset sepsis, or unexpected early discharges or transfers would make census severe retinopathy of prematurity), treatments (multiple doses prediction even more robust. These initial data provide proof of surfactant, H2 blockers, postnatal steroids) and nutritional variables principle for proceeding with models that incorporate those (average weight gain, time to full feeds) that increased the odds individual elements that can impact the reliability of census of prolonged hospital stay. We sought a predictive model using prediction.

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Conclusion Health Organization (WHO) classifications in patients with myelodysplastic syndromes: LOS prediction is improved by accounting for severity of illness results of a single-center analysis. Ann Hematol 2006; 85(8): 502–513. 11 Valeur N, Clemmensen P, Grande P, Saunama¨ki K, Investigators D. Prognostic in the first week of life, beyond factors known at birth. Models evaluation by clinical exercise test scores in patients treated with primary percutaneous including MAIN outperformed those with SNAPPE in all coronary intervention or fibrinolysis for acute myocardial infarction. Am J Cardiol populations. Our findings of poor LOS prediction for individuals, 2007; 100(7): 1074–1080. and moderately accurate LOS prediction for populations, are 12 Yeh T, Pollack M, Ruttimann U, Holbrook P, Fields A. Validation of a physiologic consistent with reports in the adult and neonatal literature. stability index for use in critically ill infants and children. Pediatr Res 1984; 18(5): Within our study populations, the difference between observed and 445–451. 13 Pollack M, Ruttimann U, Getson P. Pediatric risk of mortality (PRISM) score. Crit Care expected LOS varied less when neonates who died or transferred Med 1988; 16(11): 1110–1116. were excluded. This may be improved further by modeling 14 Klem S, Pollack M, Glass N, Spohn W, Kanter R, Zucker A et al. Resource use, categorical diagnoses (for example, necrotizing enterocolitis, efficiency, and outcome prediction in pediatric intensive care of trauma patients. sepsis) and interventions (for example, surgery) after the first J Trauma 1990; 30(1): 32–36. week of life. Prospective comparison of MAIN and SNAPPE using 15 Cockburn F, Cooke R. The CRIB (clinical risk index for babies) score: a tool for assessing initial neonatal risk. Lancet 1993; 342(8865): 193–198. more recent, inclusive, multiyear data sets is warranted. With 16 Gray J, Richardson D, McCormick M, Workman-Daniels K, Goldmann D. Neonatal recalibration of prediction equations, and validation of individual therapeutic intervention scoring system: a therapy-based severity-of-illness index. LOS predictions, modeling NICU census may yet be realized. Pediatrics 1992; 90: 561–567. 17 Verma A, Weir A, Drummond J, Mitchell B. Performance profile of an outcome measure: morbidity assessment index for newborns. J Epidemiol Community Health Conflict of interest 2005; 59: 420–426. The authors declare no conflict of interest. 18 Escobar G, Fischer A, Li D, Kremers R, Armstrong M. Score for neonatal acute physiology: validation in three kaiser permanente neonatal intensive care units. Pediatrics 1995; 96: 918–922. Acknowledgments 19 Verma A, Okun N, Maguire T, Mitchell B. Morbidity Assessment Index for Newborns: a composite tool for measuring newborn health. Am J Obstet Gynecol 1999; 181: This study was funded by the Department of Pediatrics, Women & Infants’ 701–708. Hospital of Rhode Island. 20 Richardson D, Gray J, McCormick M, Workman K, Goldmann D. Score for neonatal acute physiology: a physiologic severity index for neonatal intensive care. Pediatrics References 1993; 91: 617–623. 21 Richardson D, Corcoran J, Escobar G, Lee S. SNAP-II and SNAPPE-II: simplified 1 Knaus W, Zimmerman J, Wagner D, Draper E, Lawrence D. APACHE-acute physiology newborn illness severity and mortality risk scores. J Pediatr 2001; 138: 92–100. and chronic health evaluation: a physiologically based classification system. Crit Care 22 Shulman J. Studying determinants of length of hospital stay. J Perinatology 2006; 26: Med 1981; 9(8): 591–597. 243–245. 2 LeGall J, Loirat P, Alperovitch A. A simplified acute physiology score for ICU patients. 23 Nakagawa S, Freckleton R. Missing inaction: the dangers of ignoring missing data. Crit Care Med 1984; 12: 975–977. Trends Ecol Evol 2008; 23(11): 592–596. 3 Lemeshow S, Teres D, Avrunin J. Refining intensive care unit outcome prediction by 24 Akaike H. A new look at the statistical model identification. IEEE Transact Automatic using changing probabilities of mortality. Crit Care Med 1988; 16. Control 1974; 19(6): 716–723. 4 Herridge M. Prognostication and intensive care unit outcome: the evolving role of 25 Fanaroff AA, Stoll BJ, Wright LL, Carlo WA, Ehrenkranz RA, Stark AR et al. Trends in scoring systems. Clin Chest Med 2003; 24: 751–762. neonatal morbidity and mortality for very low birthweight infants. Am J Obstet Gynecol 5 Rafkin H, Hoyt J. Objective data and quality assurance programs. Current and future (2007196); 147: e141–e147 .e148. trends. Crit Care Clin 1994; 10(1): 157–177. 26 Meadow W, Reimshisel T, Lantos J. Birth weight-specific mortality for extremely low 6 Zupancic J, Richardson D. Characterization of the triage process in neonatal intensive birth weight infants vanishes by four days of life: epidemiology and ethics in the care. Pediatrics 1998; 102: 1432–1436. neonatal intensive care unit. Pediatrics 1996; 97(5): 636–643. 7 Glance L, Osler T, Shinozaki T. Intensive care unit prognostic scoring systems to 27 Singh Jaideep, Lantos John, Meadow William. End-of-life after birth: death and dying predict death: a cost-effectiveness analysis. Crit Care Med 1998; 26(11): 1842–1849. in a neonatal intensive care unit. Pediatrics 2004; 114: 1620–1626. 8 Garcıá S, Ruza F, Alvarado F, Madero R, Delgado M, Dorao P et al. Analysis of costs in 28 Bannwart D, Rebello C, Sadek L, Pontes M, Ramos J, Leone C. Prediction of length a pediatric ICU. Intensive Care Med 1997; 23(2): 218–225. of hospital stay in neonatal units for very low birthweight infants. J Perinatology 9 Zimmerman J, Kramer A, McNair D. Intensive care unit length of stay: benchmarking 1999; 19(2): 92–96. based on acute physiology and chronic health evaluation (APACHE) IV*. Crit Care 29 Cotten C, Oh W, McDonald S, Carlo W, Fanaroff A, Duara S et al. Prolonged Med 2006; 34: 2517–2529. hospital stay for extremely premature infants: risk factors, center differences, and the 10 Mu¨ller-Berndorff H, Haas P, Kunzmann R, Schulte-Mo¨nting J, Lu¨bbert M. Comparison impact of mortality on selecting a best-performing center. J Perinatology 2005; 25: of five prognostic scoring systems, the French-American-British (FAB) and World 650–655.

Supplementary Information accompanies the paper on the Journal of Perinatology website (http://www.nature.com/jp)

Journal of Perinatology Journal of Perinatology (2013) 33,87–93 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Physical activity and maternal–fetal circulation measured by Doppler ultrasound

NC Nguyen1,2, KR Evenson1, DA Savitz3, H Chu4, JM Thorp5 and JL Daniels1 1Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Department of Research and Training, Hanoi Obstetrics and Gynecology Hospital, Hanoi, Vietnam; 3Division of Biological and Medical Sciences, Department of Community Health Epidemiology Section, and Department of Obstetrics and Gynecology, Brown University, Providence, RI, USA; 4Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN, USA and 5Department of Obstetrics and Gynecology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

associated with adverse fetal and maternal outcomes.2–4 Regular Objective: To examine the association of physical activity on maternal– physical activity and exercise during pregnancy may alter the fetal circulation measured by uterine and umbilical artery Doppler flow maternal hemodynamic system and placental function by velocimetry waveforms. increasing placental volumes and growth rates.5,6 Enhanced Study Design: Participants included 781 pregnant women with Doppler placental growth and vascularity may be an adaptive response to ultrasounds of the uterine and umbilical artery and who self-reported past intermittent reductions in feto-placental blood flow during 7 week physical activity. Linear and generalized estimating equation exercise. It has been hypothesized that these effects may increase regression models were used to examine these associations. the rate of placental perfusion and transfer function and reduce the fraction of non-functional tissue in the placenta,8,9 which might be Result: Moderate-to-vigorous total and recreational activity were associated beneficial for both the mother and the fetus.7 with higher uterine artery pulsatility index (PI) and an increased risk of Doppler ultrasound provides a non-invasive way to examine uterine artery notching as compared with reporting no total or recreational placental blood flow and placental vascular resistance on the maternal physical activity, respectively. Moderate-to-vigorous work activity was and fetal sides of the placenta, enabling information about the associated with lower uterine artery PI and a reduced risk of uterine artery maternal–fetal circulation.10,11 For example, when placental notching as compared with no work activity. No associations were identified impedance increases, the uterine and umbilical arterial diastolic flow with the umbilical circulation measured by the resistance index. decreases, which is associated with persistence of a diastolic notch in Conclusion: In this epidemiologic study, recreational and work activity uterine arteries or with either low, absent or even reversed end-diastolic were associated with opposite effects on uterine artery PI and uterine blood flow in the umbilical artery that can be detected by Doppler artery notching, although associations were modest in magnitude. ultrasound.10,12,13 Numerous studies have established an association Journal of Perinatology (2013) 33, 87–93; doi:10.1038/jp.2012.68; between abnormal Doppler velocimetry waveforms in the uterine published online 7 June 2012 and umbilical arteries and adverse pregnancy outcomes such as Keywords: work; recreational activity; maternal–fetal blood flow; pre-eclampsia, intrauterine growth retardation, and prediction of fetal pregnancy; Doppler flow velocimetry waveform; pre-eclampsia distress, perinatal mortality, or admission to the neonatal intensive care unit, but the association is weaker in low-risk pregnant women.10,13–18 Several randomized clinical trials have evaluated the immediate Introduction effect of maternal exercise on maternal–fetal circulation with Doppler The American College of Obstetricians and Gynecologists velocimetry, although the sample sizes have been small. Most encourages pregnant women, in the absence of either medical or measurements were taken before and soon after exercise. Findings for the uterine artery Doppler indices have not been consistent, including obstetric complications, to engage in regular, moderate intensity 19–21 1 studies finding no meaningful changes, an increase in the exercise. This recommendation is based on scientific evidence that 22–24 moderate intensity exercise during pregnancy is not generally indices and an increase followed by a decrease in the indices following bouts of exercise.25,26 More consistently, studies have found Correspondence: Dr KR Evenson, UNC Department of Epidemiology, Bank of America Center, no change in umbilical artery Doppler indices with exercise.19,22–24 137 East Franklin Street, Suite 306, Chapel Hill, NC 27514, USA. These studies were conducted on small samples; thus, no general E-mail: [email protected] Received 7 November 2011; revised 4 April 2012; accepted 7 May 2012; published online 7 June conclusion has been made regarding the acute effect of physical 2012 exercise on resistance of maternal–fetal blood flow. Physical activity and maternal–fetal circulation NC Nguyen et al 88

We are not aware of any observational studies that report on In this analysis, we focused on total activity and two typical types associations between physical activity during pregnancy and of physical activity (work and recreational activity) because national changes of Doppler flow measurements of the uteroplacental unit. guidelines or recommendations focus on these specific types of This study provides a comprehensive analysis of overall physical activities.1,30 To address perceived or relative intensity, for each type activity and several components of physical activity on maternal– of activity we calculated the total number of hours per week overall fetal circulation during mid-pregnancy. and in ‘fairly light’, ‘somewhat hard’ and ‘hard or very hard’ intensity (the latter two defines moderate-to-vigorous intensity). To examine absolute intensity, we multiplied the hours per week in Methods each activity by its corresponding MET value and summed them up Study population to calculate total MET-hours per week and MET-hours per week for This analysis includes participants in the third phase of the PIN moderate-to-vigorous activity, defined as activities with a MET value (Pregnancy, Infection and Nutrition) Study, a prospective study X4.8, which corresponds to the lower range of moderate intensity 31 examining etiologic factors (physical activity, stress and placental for women 20 to 39 years of age. Total activity was calculated by vascular compromise) for preterm delivery. The study protocol was summing up all the values for all activity types. Each physical approved by the Institutional Review Board at the University of activity variable in the analysis was categorized into four levels: (1) North Carolina, Chapel Hill, NC, USA. Women seeking services no activity, (2) fairly light activity, (3) moderate-to-vigorous activity before 20 weeks’ gestation from prenatal clinics at the University of less than or equal to the median value among participants that North Carolina Hospitals between January 2001 and June 2005 were reported that activity and (4) moderate-to-vigorous activity greater asked to participate with written informed consent. Exclusion than the median value. The only exception was for moderate-to- criteria included being less than age 16, non-English speaking, not vigorous work activity, which had a low prevalence, so we collapsed planning to continue care or deliver at the study site, carrying the variable into three categories. multiple gestations, or not having a telephone for the phone interviews. During the course of pregnancy, the women were asked Outcome measurement to complete two research clinic visits and two telephone interviews. During the first clinic visit, at 15 to 20 weeks’ gestation, the After excluding women who enrolled in the study a second or third sonographer performed Doppler ultrasonography to obtain flow time, women who did not complete Doppler ultrasounds of either or velocity waveforms in the uterine arteries (left and right). During both of uterine and umbilical artery at two clinic visits, or who did the second clinic visit, at 24 to 29 weeks’ gestation, Doppler not complete the first telephone interview with information from a ultrasonography was similarly performed in the uterine arteries, as 1-week physical activity recall, leaving 781 women in the analysis. well as the umbilical artery. The woman lay in a semi-recumbent position, and both uterine arteries were examined at their crossing Physical activity measurement with the external iliac artery using a 3.5 MHz transabdominal A 1-week recall of physical activity was obtained from the phone probe. For the umbilical artery, Doppler velocity waveforms were interview at 17 to 22 weeks’ gestation. The questionnaire included obtained from the free-floating loop of the umbilical cord during six separate sections for work, recreational, outdoor household, fetal quiescence. Doppler waveform indices were calculated from indoor household, child–adult care and transportation, with computerized planimetry as systolic/diastolic ratio, pulsatility index evidence for validity and reliability reported elsewhere.27 The (PI) in the uterine artery measures, and as resistance index (RI) in questionnaire assessed frequency and duration of all moderate and the umbilical artery measures. Also, presence of an early diastolic vigorous physical activities the women participated in during the notch in the uterine arteries or umbilical artery, presence and past week. For example, a question regarding participation in direction of diastolic flow in umbilical artery, and location of the particular modes of recreational activity stated: ‘in the past-week, placenta, categorized as anterior, posterior, left lateral, right lateral, did you participate in any non-work, recreational activity or previa and fundal in the uterine cavity were recorded. exercise, such as walking for exercise, swimming, dancing, that For the uterine artery analysis, PI was chosen as a continuous caused at least some increase in breathing and heart rate?’ If the outcome measure, because of its nearly normal distribution and woman answered ‘Yes’, the interviewer asked her to list all types of having very few outliers. The mean of the left and right artery PI recreational activities. For each type of activity, the woman reported measurements was used for the final analysis. We also used a the number of sessions in the past week, duration of each session dichotomous variable that indicated the presence of an early and her perception of the intensity classified as: ‘fairly light’, diastolic notch in either or both the left and right uterine arteries. ‘somewhat hard’ and ‘hard or very hard’, from the Borg scale.28 For the umbilical artery analysis, RI was chosen as a For scoring based on absolute intensity, we assigned an intensity continuous outcome measure because of its nearly normal code for each activity using metabolic equivalent (MET) from distribution in the study population. With very few participants with published tables.29 adverse umbilical artery outcomes, such as notch present or

Journal of Perinatology Physical activity and maternal–fetal circulation NC Nguyen et al 89

Table 1 Doppler waveform indices used in this study

Arteries examined Outcome indices Time of measurement Calculation

Uterine arteries Mean of PI Measured twice at 15–20 and 24–29 PI ¼ (S–D)/A weeks’ gestation Any early diastolic notch in either or both Measured twice at 15–20 and 24–29 Yes or no uterine arteries weeks’ gestation Umbilical artery RI Measured once at 24–29 weeks’ gestation RI ¼ (S–D)/S

Abbreviations: A, time-averaged maximum velocity; D, end-diastolic velocity; PI, pulsatility index; RI, resistance index; S, peak systolic velocity. reversed diastolic flow in the umbilical artery Doppler, we did not continuous variable (‘other physical activity’) defined by analyze these measures. A summary of Doppler waveform indices subtracting of the specific exposure activity value of interest from used in this study are presented in Table 1. the total activity value in the final models. We reported corresponding regression coefficients and P-values for physical Covariate measurement activity variables in each model. Other information on participants was obtained from the first We fit generalized estimating equation models to the uterine phone interview at 17 to 22 weeks’ gestation and medical records. artery Doppler data using PROC GENMOD with REPEATED Sociodemographic variables included age at enrollment, race statement in SAS (SAS Institute, Cary, NC, USA) for the continuous (white, black, other), marital status, completed years of education outcome as mean uterine PI, measured twice.35 We reported beta and household percentage of poverty for 2001.32 Pre-pregnancy regression coefficients with 95% confidence intervals (CIs) and P- body mass index was calculated from self-reported weight and values for physical activity variables in each model. The betas had measured height (kg m–2) and categorized based on the Institute an interpretation as the association of a one-level change in each of Medicine guidelines.33 Adequacy of total weight gain was exposure on mean uterine PI as a continuous outcome, controlling calculated as a ratio of observed weight to expected weight based on for all other covariates and adjusting for the longitudinal the Institute of Medicine guidelines in effect during the time of clustering. Also using generalized estimating equation models for data collection.33 Pregnancy and medical history variables included the presence of a notch in either or both uterine arteries, as a reproductive history (parity, history of stillbirth, miscarriage and dichotomous outcome, measured twice, we reported crude and abortions), chronic hypertension, pre-existing diabetes and adjusted relative risk with their CIs. All final models were adjusted bleeding during pregnancy. Lifestyle behaviors included for appropriate confounders (that is, removal of the variable did prepregnancy body mass index, smoking, alcohol consumption and not change the physical activity exposure estimate by >10%), drug use. All of these covariates were explored as potential gestational age at the first and second clinic visit, placental confounders in each of the models. Gestational age at the location at the second visit and ‘other physical activity’. ultrasound measurements was included in all models. Reproductive history (parity, history of stillbirths, miscarriages and abortions), bleeding during pregnancy, prepregnancy body mass Statistical analyses index, adequacy of maternal weight gain, alcohol use and drug use Multiple linear regression modeling was performed for the were not confounders in any model, and thus are not adjusted for umbilical RI and started with full models, including physical in the multivariable analyses. Pregnancy outcomes (for example, activity, all covariates as previously described, gestational age at the pre-eclampsia, preterm birth), which were evaluated at the time of ultrasound and placental location. We used a backward termination of pregnancy were not included in the analysis as elimination approach to remove variables from the model.34 Two confounders or effect modifiers. criteria were used for elimination of a variable: (1) P-value for the t-test for the presence of this variable in the model exceeding 0.10, indicating the variable did not contribute meaningfully to the Results prediction of umbilical RI, and (2) removal of the variable from Descriptive characteristics the model not changing the estimate of coefficients for physical Distribution of sociodemographic, lifestyle and pregnancy activity exposure measure by >10%, indicating the variable was characteristics of study participants are presented in Table 2. not a confounder. We retained any confounder identified in any of Women were mostly white, married and well educated. Almost 40% the three physical activity models in all final models, together with of women were overweight or obese. At 17 to 22 weeks’ gestation, gestational age at the second ultrasound measure. Finally, to 30% of women reported at least some work-related physical activity account for other types of physical activity when examining the in the past week and 63% reported at least some recreational association with work and recreational activity, we included a physical activity.

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Table 2 Selected sociodemographic, lifestyle, and pregnancy characteristics of found regarding regular exercise before pregnancy, and during the the study participants (N ¼ 781) first and second trimester of pregnancy. However, women who were Number Percent included in the analysis were more likely to have a lower percent below poverty than women not included in the analysis. Age in years p20 76 9.7 Physical activity and uterine artery PI and early diastolic 21–34 596 76.3 notching X35 109 14.0 At the first clinic visit, the mean uterine PI was 1.15. (s.d. ¼ 0.46), and 29% had an early diastolic notch present in either Race or both uterine arteries. At the second clinic visit, the mean uterine White 548 70.2 PI was 0.88 (s.d. ¼ 0.28), 11% had a notch present in either Black 161 20.6 or both uterine arteries, and the mean umbilical RI was 0.66 Other 72 9.2 (s.d. ¼ 0.06). Marital status Table 3 displays the generalized estimating equation models for Not married 226 28.9 the relationship between perceived intensity of physical activity and the two outcomes, mean uterine PI and any uterine artery Education in years notching at either or both first and second clinic visit. Moderate-to- High school or less (p12) 181 23.2 vigorous total and recreational activity above the median were both Some college (13–15) 162 20.7 associated with higher uterine PI as compared with no total or College graduation 438 56.1 recreational physical activity, respectively, (beta coefficient ¼ 0.092 (95% CI 0.006, 0.178) and 0.073 (95% CI 0.003, 0.144)). Moderate- Percentage of the poverty line based on to-vigorous work activity was associated with lower uterine PI (beta household income coefficient ¼0.107 (95% CI 0.164, 0.049) as compared <100 120 16.1 with reporting no work activity. The risk of any uterine artery 100–200 100 13.4 >200 526 70.5 notching was higher among women reporting moderate-to- vigorous recreational activity above the median compared with Body mass index before pregnancy those reporting no recreational physical activity (relative risk Underweight <19.8 kg m–2 104 13.5 adjusted ¼ 1.75 (95% CI 1.11 to 2.75)). Normal: 19.8– <26.0 kg m–2 369 48.0 Overweight: 26– <29.0 kg m–2 88 11.4 Physical activity and umbilical RI Obese: X29.0 kg m–2 208 27.1 The multiple linear regression models between physical activity and the umbilical RI are described in Table 4. Total, recreational and Chronic hypertension 67 9.0 work physical activity at any level of intensity were not associated with the umbilical artery RI. Pre-existing diabetes 36 4.8 Sensitivity analysis Smoking All results presented in Tables 3 and 4 were recalculated using Non-smoker 492 69.2 absolute intensity (in MET-hours per week) for physical activity, Past smoker 104 14.6 rather than perceived intensity (in hours per week). The findings Current smoker 115 16.2 were quite similar (data not shown). We also explored all recreational and work models without further adjustment for ‘other physical activity’ and our interpretations did not meaningfully We compared characteristics of women who were included in change (data not shown). the analysis (n ¼ 781) and those who were eligible, but were not included (n ¼ 1094) because of lack of information on Doppler ultrasound or 1-week recall physical activity (data not shown). Chi-square statistics were used for comparison. We found no Discussion differences between the included and excluded groups (P>0.05) on To our knowledge, this is the first epidemiologic study to most characteristics (for example, age, race, education, marital investigate the association between physical activity and vascular status, general health, pre-pregnancy body mass index, parity, resistance in the uterine and umbilical artery blood flow. We found maternal weight gain, adequacy of prenatal care and smoking). that moderate-to-vigorous total and recreational physical activity Also, no differences between the included and excluded groups were above the median was associated with a higher uterine artery PI

Journal of Perinatology Physical activity and maternal–fetal circulation NC Nguyen et al 91

Table 3 The associations between self-reported physical activity (using perceived intensity at 17–22 weeks’ gestation) and mean uterine artery PI and the risk of any uterine artery notching at both Doppler measurements (N ¼ 781).

Mean uterine artery PIa Risk of any uterine artery notching

N % Beta 95% CI of beta P-value RR unadjusted (95% CI) RR adjusted (95% CI)b

Total physical activity (median ¼ 2.75 hours per week) No activityc 52 6.6 Reference 1.00 1.00 Fairly light total activity 224 28.7 0.063 (0.023, 0.149) 0.15 0.87 (0.48, 1.57) 1.09 (0.54, 2.22) MV total activity p the median 256 32.8 0.059 (0.026, 0.143) 0.18 0.84 (0.46, 1.50) 1.23 (0.61, 2.49) MV total activity > the median 249 31.9 0.092 (0.006, 0.178) 0.04 1.01 (0.56, 1.81) 1.48 (0.73, 2.99)

Recreational activity (median ¼ 2.0 hours per week) No recreational activityc 293 37.5 Reference 1.00 1.00 Fairly light recreational activity 185 23.7 0.038 (0.026, 0.102) 0.25 1.11 (0.76, 1.62) 1.25 (0.82, 1.92) MV recreational activity p the median 175 22.4 0.045 (0.019, 0.109) 0.17 1.21 (0.82, 1.78) 1.43 (0.93, 2.18) MV recreational activity > the median 128 16.4 0.073 (0.003, 0.144) 0.04 1.59 (1.04, 2.39) 1.75 (1.11, 2.75)

Work activity No work activityc 546 69.9 Reference 1.00 1.00 Fairly light work activity 146 18.7 0.014 (0.075, 0.046) 0.64 1.00 (0.69, 1.44) 1.19 (0.80, 1.77) Some MV work activity 89 11.4 0.107 (0.164, 0.049) p0.001 0.61 (0.39, 0.98) 0.64 (0.38, 1.08) Abbreviations: CI, confidence interval; MV, moderate to vigorous; PI, pulsatility index; RR, relative risk. aAdjusted for household percentage of poverty (<100, 100–200, >200), pre-existing diabetes (yes, no), smoking (no, past, current), gestational age at first and second visit (weeks) and other physical activity. bAdjusted for household percentage of poverty (<100, 100–200, >200), pre-existing diabetes (yes, no), smoking (no, past, current), gestational age at first and second visit (weeks), placental location at second visit (anterior, posterior, other) and other physical activity. cReferent group, women with no specific activity. Median among participants who reported at least some moderate-to-vigorous physical activities. In all models, variables with more than two levels were coded as indicator variables. and notching, which was in agreement with previous randomized Among women engaging in exercise during pregnancy, many other trials assessing acute exercise response.22–24 In contrast, we biological effects are observed including increased resting maternal observed a lower uterine artery PI among women reporting plasma volume, intervillous space blood volume, enhanced feto- moderate-to-vigorous work activity, suggesting a reduction of placental growth, placental function and vascularity.5–7 impedance in uterine circulation. We can only speculate as to the Observations in the placental biopsy after delivery reveal a reason for the differences in associations between recreational and significantly greater total vascular volume and total capillary work activities; it may be that recreational activities by nature are volume in the placental villi among women engaging in exercise.8 shorter term and episodic in contrast to work activities, which may Other important findings in the placental biopsy included increased occur throughout the day. total villous surface area, increased cell proliferation and reduced We found an increased risk of the presence of any uterine artery fraction of non-functional tissue.8,37 The chronic effects on these notching among women reporting moderate-to-vigorous physiological and functional changes increase overall placental recreational activity above the median, and somewhat higher for perfusion, placental bed blood flow at rest, and both oxygen and total physical activity, suggesting somewhat higher chronic substrate delivery.7,8 All of these changes imply a reduction of impedance of uterine artery circulation. However, we also found a resistance in the placental blood flow among women engaging in reduced risk of uterine artery notching among women reporting exercise during pregnancy, consistent with the apparent reduction moderate-to-vigorous work activity. in resistance associated with work activity but not to our total and A link between physical activity and increased resistance in the recreational activity findings. uteroplacenta is plausible biologically. In normal pregnancy, Our study found no associations between types of physical maternal hemodynamic adaptations, including changes in activity, including work and recreational activity, at any level of vascular tone, increase in placental bed blood flow through intensity with the umbilical Doppler RI. Umbilical resistance hormonally mediated vascular growth and remodeling and reflects fetal circulation and our findings support the null decreased adrenergic response, lead to low vascular impedance in observations of other randomized trials assessing acute response of the uteroplacenta and decreased placental vascular resistance.9,11,36 exercise.19,22–24

Journal of Perinatology Physical activity and maternal–fetal circulation NC Nguyen et al 92

Table 4 The association between past week physical activity (using perceived misclassiﬁcation of exposure is likely to be random with respect to intensity at 17–22 weeks of pregnancy) and umbilical artery resistance index at the outcomes and therefore, is most likely to bias relationships 24–29 weeks of pregnancy (N ¼ 781) toward the null. Doppler velocimetry indices were also subject to Modeling with physical activity in Beta (95% CI) P-value some variances between and within ultrasonographers. The perceived intensitya ﬁndings could also be biased by inadequate adjustment for unmeasured confounding such as nutrition status or stress during Total physical activity (median ¼ 2.75 pregnancy. Timing between measures of physical activity and the hours per week) Doppler ultrasound exams may introduce some biases because of No activityb Reference variable patterns of physical activity during pregnancy. Fairly light total activity 0.005 (0.013, 0.021) 0.62 MV total activity p the median 0.009 (0.011, 0.027) 0.39 MV total activity > the median 0.007 (0.014, 0.019) 0.50 Conclusion Recreational activity (median ¼ 2.0 hours In this epidemiologic study, recreational and work activity were per week) b associated with opposite effects on uterine artery PI and uterine No recreational activity Reference artery notching, although associations were modest in magnitude. Fairly light recreational activity 0.002 (0.021, 0.009) 0.79 No association was found between physical activity and umbilical MV recreational activity p the median 0.002 (0.016, 0.017) 0.71 MV recreational activity > the median 0.004 (0.017, 0.011) 0.58 circulation measured by the RI. Future studies could consider a time series design with multiple prospective measurements of both Work activity physical activity and Doppler indices during pregnancy. Other No work activityb Reference parameters, such as middle cerebral artery Doppler indices, could Fairly light work activity 0.005 (0.013, 0.006) 0.43 be included in future studies. Some MV work activity 0.001 (0.010, 0.014) 0.86

Abbreviations: CI, confidence interval; MV, moderate to vigorous. aAdjusted for household percentage of poverty (<100, 100–200, >200), weight gain ratio Conflict of interest of observed weight/expected weight based on IOM guidelines (<1, 1– <2, X2), gestational age at second visit (weeks), placental location at second visit (anterior, The authors declare no conflict of interest. posterior, other) and other physical activity. bReferent group, women with no specific activity. Median among participants who reported at least some moderate-to-vigorous activities. In all models, variables with more than two levels were coded as indicator variables. Acknowledgments This study was funded by the following grants: National Institutes of Health (NIH)/ National Institute of Child Health and Human Development #HD37584, NIH/General Strengths and limitations Clinical Research Center #RR00046 and NIH/National Cancer Institute In this study, physical activity was collected through a 1-week #R01CA109804. Dr Nguyen was funded in part by a grant from the Vietnam recall questionnaire to capture comprehensive information on the Education Foundation (VEF). The content is solely the responsibility of the authors type, frequency, duration and intensity of all types of physical and does not necessarily represent the official views of the NIH or VEF. The activity during pregnancy. We assessed the intensity of each activity Pregnancy, Infection and Nutrition Study, along with the ancillary projects, is a joint using both perceived (self-reported) and absolute intensity (from effort of many investigators and staff members whose work is gratefully acknowledged. the compendium of physical activities).29 Outcome data were measured at one or two different time points during pregnancy, taking into account the variability of these measures along with References gestational age. Information on key confounders was collected in 1 ACOG. Exercise during pregnancy and the postpartum period. ACOG Committee detail, allowing us to evaluate the effects of specific types of Opinion No. 267. 2002. Obstet Gynecol 2002; 99: 171–173. physical activity and we were able to control for other activities 2 Artal R, O’Toole M, White S. Guidelines of the American College of Obstetricians and Gynecologists for exercise during pregnancy and the postpartum period. Br J Sports simultaneously. Med 2003; 37: 6–12. However, several limitations of this study should be noted. 3 Dempsey J, Butler C, Williams M. No need for a pregnant pause: physical activity may Although there is evidence to support the validity and reliability of reduce the occurrence of gestational diabetes mellitus and preeclampsia. Exercise Sport the physical activity questionnaire among pregnant women,27 Sci Rev 2005; 33: 141–149. measurement error is inevitable based on of self-reports. Physical 4 Paisley TS, Joy EA, Price Jr RJ. Exercise during pregnancy: a practical approach. activity can be variable over a short period of time, so that physical Current Sports Med Reports 2003; 2: 325–330. 5 Clapp III JF, Kim H, Burciu B, Schmidt S, Petry K, Lopez B. Continuing regular activity measured in a 1-week period may not be representative exercise during pregnancy: effect of exercise volume on fetoplacental growth. Am J over a longer interval, especially during pregnancy. Such Obstet Gynecol 2002; 186: 142–147.

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6 Pivarnik JM, Mauer MB, Ayres NA, Kirshon B, Dildy GA, Cotton DB. Effects of chronic 21 Steegers EA, Buunk G, Binkhorst RA, Jongsma HW, Wijn PF, Hein PR. The influence of exercise on blood volume expansion and hematologic indices during pregnancy. maternal exercise on the uteroplacental vascular bed resistance and the fetal heart rate Obstet Gynecol 1994; 83: 265–269. during normal pregnancy. Eur J Obstet Gynecol Reprod Biol 1988; 27: 21–26. 7 Clapp III JF. The effects of maternal exercise on fetal oxygenation and feto-placental 22 Erkkola RU, Pirhonen JP, Kivijarvi AK. Flow velocity waveforms in uterine and growth. Eur J Obstet Gynecol Reprod Biol 2003; 110(Suppl 1): S80–S85. umbilical arteries during submaximal bicycle exercise in normal pregnancy. Obstet 8 Jackson MR, Gott P, Lye SJ, Ritchie JW, Clapp III JF. 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The effect of maternal exercise on uterine artery velocimetry S (eds). A Colour Atlas of Doppler Ultrasonography in Obstetrics. An Introduction to waveforms. J Obstet Gynaecol 1998; 18: 14–17. its Use in Maternal Fetal Medicine. Hodder Arnold Publications: London, 2000 pp 26 Rafla NM. Umbilical artery flow velocity waveforms following maternal exercise. 677–712. J Obstet Gynaecol 1999; 19: 385–389. 12 Axt-Fliedner R, Schwarze A, Nelles I, Altgassen C, Friedrich M, Schmidt W et al. The 27 Evenson KR and Wen F. Measuring physical activity in pregnant women using a structured value of uterine artery Doppler ultrasound in the prediction of severe complications in one-week recall questionnaire: evidence for validity and reliability. Intl J Behavioral Nutr a risk population. Arch Gynecol Obstet 2005; 271: 53–58. Physical Activity 2010; 7: 21. Available at http://www.ijbnpa.org/content/7/1/21. 13 Bower S, Schuchter K, Campbell S. 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Effects of Work on Pregnancy 1999, Accessed 7 as a predictor of adverse pregnancy outcome in high-risk women. Ultrasound Obstet November 2011 at http://www.ama-assn.org/ama1/pub/upload/mm/443/csaa-99.pdf. Gynecol 2000; 15: 7–12. 31 Pollock M, Gaesser G, Butcher J, Despres J, Dishman R, Franklin B et al. American 16 Irion O, Masse J, Forest JC, Moutquin JM. Prediction of pre-eclampsia, low birthweight College of Sports Medicine Position Stand: the recommended quantity and quality of for gestation and prematurity by uterine artery blood flow velocity waveforms analysis exercise for developing and maintaining cardiorespiratory and muscular fitness, and in low risk nulliparous women. Br J Obstet Gynaecol 1998; 105: 422–429. flexibility in healthy adults. Med Sci Sport Exer 1998; 30: 975–991. 17 Todros T, Ronco G, Fianchino O, Rosso S, Gabrielli S, Valsecchi L et al. Accuracy of the 32 Proctor B, Dalaker J. US Bureau of the Census, Current Population Reports. 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Journal of Perinatology Journal of Perinatology (2013) 33, 107–111 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Maternal ABO blood group and adverse pregnancy outcomes

C Phaloprakarn and S Tangjitgamol Department of Obstetrics and Gynecology, Faculty of Medicine Vajira Hospital, University of Bangkok Metropolis, Bangkok, Thailand

both A (glycosyltransferase a1,3-GalNAc-transferase) and B Objective: To determine the relationship between maternal ABO blood (a1,3Gal-transferase) antigens.3,4 The O allele encodes an inactive group and risk of adverse pregnancy outcomes. glycosyltransferase, hence neither A nor B antigen is synthesized.3 Study Design: Data on ABO phenotypes and pregnancy outcomes were Both A and B antigens are found on the surface of red blood cells collected from medical records of 5320 singleton pregnant women who had and other tissues including vascular endothelium, epidermis and 5 ABO blood testing and follow-up care until delivery in our institution. glandular or other epitheliums lining visceras. Adverse pregnancy outcomes that were studied in relation to maternal blood Aside from its physiologic role, recent studies have shown group included preeclampsia, gestational diabetes mellitus (GDM), preterm associations between particular ABO phenotypes and various delivery, low birth weight (LBW) and small for gestational age (SGA) infants. pathologic events, for example, infection, bleeding disorder, thromboembolic disease, cancers and so on.6–9 The possible Result: Out of 5320 women, 350 (6.6%), 333 (6.3%) and 543 (10.2%) mechanisms for the associations are the influences of some genetic women were diagnosed with preeclampsia, GDM and preterm delivery, variants at the ABO locus on the aberrations of some biological respectively. LBW and SGA were, respectively, observed in 394 (7.4%) and substances, such as proinflammatory cytokine, adhesion molecules 178 (3.3%) infants. By uni- and multivariable analyses, women with A or and thrombogenic factor.10,11 The effects of blood types on AB blood types, but not B, were found at increased risk of preeclampsia pregnancy outcomes have also been reported, however, with compared with O type individuals; adjusted relative risks were 1.7 (95% inconsistent results.12–15 Some authors found a greater risk of confidence interval (CI), 1.3 to 2.3; P ¼ 0.001) for A phenotype and 1.7 preeclampsia in women with A or AB blood types,12–14 whereas (95% CI, 1.1 to 2.6; P ¼ 0.01) for AB phenotype. There were no significant another study found that percentage of O blood type was high relationships between blood types and GDM, preterm delivery, LBW or SGA. among women with gestational diabetes mellitus (GDM).16 Others Conclusion: Maternal ABO blood group was associated with the risk of did not find any relationship between maternal blood group and preeclampsia, but not with GDM, preterm delivery, LBW or SGA. pregnancy outcomes including preeclampsia, postpartum Journal of Perinatology (2013) 33, 107–111; doi:10.1038/jp.2012.73; hemorrhage, intrauterine growth restriction, large for gestational published online 7 June 2012 age (GA) infants or stillbirth.15 Bearing in mind the limited data from a few studies with Keywords: ABO blood group; pregnancy outcomes; risk inconsistent results, additional information is needed. Furthermore, all previous studies were carried out in Western populations with Introduction possibly different distributions of ABO phenotypes from Asian women. The ABO blood group is the first and the most well-known blood Hence, more research on this subject is necessary. The aim of this type system in humans.1 This blood system has important roles in study was to evaluate the relationship between maternal ABO blood human physiology, biomedical science, anthropology, legal issues, group and risks of pregnancy complications or adverse pregnancy as well as transfusion and transplantation medicine.1,2 The blood outcomes in Thai women. More specifically, we focused on the group of an individual is controlled by a single gene (the ABO complications or adverse outcomes that were previously reported on gene) located on the long arm of chromosome 9 (9q34) with three this topic and the ones that are presently recognized as unfavorable variant alleles: A, B and O.3 The A and B alleles encode pregnancy outcomes. These included preeclampsia, GDM, preterm some specific enzymes (glycosyltransferases) that produce delivery, low birth weight (LBW) and small for GA (SGA) infants.

Correspondence: Dr C Phaloprakarn, Department of Obstetrics and Gynecology, Faculty of Medicine Vajira Hospital, University of Bangkok Metropolis, 681 Samsen Road, Dusit district, Bangkok 10300, Thailand. Methods E-mail: [email protected] Received 9 September 2011; revised 2 April 2012; accepted 7 May 2012; published online 7 June This study was approved by the Bangkok Metropolitan 2012 Administration Ethics Committee for Research Involving Human ABO blood group and pregnancy outcomes C Phaloprakarn and S Tangjitgamol 108

Table 1 Characteristic features of the study population stratiﬁed by ABO phenotypes

Characteristic Total (n ¼ 5320) Blood group P-valuea

A(n ¼ 1153) B (n ¼ 1832) AB (n ¼ 379) O (n ¼ 1956)

Age (years), n (%) <20 701 (13.2) 161 (14.0) 244 (13.3) 51 (13.4) 245 (12.5) 0.79 20–34 3922 (73.7) 832 (72.1) 1345 (73.4) 283 (74.7) 1462 (74.8) X35 697 (13.1) 160 (13.9) 243 (13.3) 45 (11.9) 249 (12.7)

Parity, n (%) Nullipara 2668 (50.2) 566 (49.1) 933 (50.9) 200 (52.8) 969 (49.5) 0.51 Multipara 2652 (49.8) 587 (50.9) 899 (49.1) 179 (47.2) 987 (50.5)

Body mass index (kg m2), n (%) <20.0 1789 (33.6) 392 (34.0) 631 (34.4) 135 (35.6) 631 (32.3) 0.49 20.0–24.9 2396 (45.0) 503 (43.6) 812 (44.3) 172 (45.4) 909 (46.5) 25.0–29.9 835 (15.7) 187 (16.2) 276 (15.1) 55 (14.5) 317 (16.2) X30.0 300 (5.7) 71 (6.2) 113 (6.2) 17 (4.5) 99 (5.0) aw2 test.

Subjects. We performed a retrospective analysis of all consecutive Statistical analysis was performed with the SPSS software Thai pregnant women who attended our antenatal clinic between 1 package version 11.5 (SPSS, Chicago, IL, USA). Continuous July 2008 and 30 June 2010. Inclusion criteria were singleton variables among the four groups of women with different blood pregnancies who sought their first antenatal care at GA p14 weeks, types (A, B, AB or O) were compared by using one-way analysis of and who underwent ABO blood typing along with other basic variance; when overall analysis was significant, the intergroup antenatal care laboratory investigations in our institution. Exclusion comparisons were then made by the Scheffe method. Categorical criteria were those who had: known underlying disease or condition variables between groups were compared with the w2 test. Owing to that could affect the pregnancy outcomes (that is, chronic its absence of both A and B antigens, O blood type was used as the hypertension, overt diabetes, renal or collagen vascular disease, reference group. The relative risks (RRs) with 95% confidence hyperthyroidism, smoking or congenital fetal anomalies), Rh- intervals (CIs) of the five outcomes studied in A, B and AB blood negative blood type, delivery elsewhere and incomplete clinical data. types were analyzed by stepwise logistic regression adjusted for Data collected were: maternal age, parity, body mass index at potential confounders. P-value <0.05 was considered statistically first visit, ABO blood group, GA at delivery, route of delivery, significant. neonatal birth weight and adverse pregnancy outcomes. ABO blood group was determined by a standard blood group serology analysis. This included both testing of A and B antigens on red blood cells Results (with anti-A and anti-B reagents) and testing of anti-A and anti-B A total of 5320 gravidas were included in this study; 50.2% were antibodies in serum (with A and B antigens). The result of each nulliparous. Mean age and body mass index at first visit were test was certified by laboratory personnel before submitting the 27.0±6.3 years and 22.2±4.3 kg m2, respectively. The final report. Pregnancy complications or adverse pregnancy distribution of ABO phenotypes in the study population was as outcomes in this study included preeclampsia, GDM, preterm follows: A ¼ 21.7%, B ¼ 34.4%, AB ¼ 7.1% and O ¼ 36.8%. Among delivery, LBW and SGA infants. Preeclampsia was defined based women with four different blood types, no significant differences on the guideline of the American College of Obstetricians and in terms of teenage or elderly mothers, rate of nullipara and Gynecologists.17 All women were screened for GDM. Those with a percentages of overweight or obesity were observed. Details of the positive screen were scheduled for a diagnostic test. The diagnosis characteristic features of the study population are presented in of GDM was based on the Carpenter and Coustan criteria.18 Table 1. Preterm delivery included a delivery before the completion of 37 Out of 5320 women, 350 (6.6%), 333 (6.3%) and 543 (10.2%) weeks of gestation. Neonatal birth weights <2500 g or under the had preeclampsia, GDM and preterm delivery, respectively. With 10th percentile for that particular GA, using a Thai infant birth regard to their babies, 394 (7.4%) and 178 (3.3%) were, weight nomogram,19 were considered LBW or SGA infants, respectively, diagnosed with LBW and SGA infants. Table 2 presents respectively. birth outcomes of the women. There were no significant differences

Journal of Perinatology ABO blood group and pregnancy outcomes C Phaloprakarn and S Tangjitgamol 109

Table 2 Birth outcomes of the study population stratiﬁed by ABO phenotypes

Outcome Total (n ¼ 5320) Blood group P-value

A(n ¼ 1153) B (n ¼ 1832) AB (n ¼ 379) O (n ¼ 1956)

Gestational age at delivery (weeks), mean (SD) 38.3 (1.7) 38.2 (1.7) 38.3 (1.7) 38.3 (1.5) 38.3 (1.6) 0.25a Route of delivery, n (%) 0.25b,c Cesarean route 1593 (29.9) 346 (30.0) 540 (29.5) 124 (32.7) 583 (29.8) Vaginal route 3727 (70.1) 807 (70.0) 1292 (70.5) 255 (67.3) 1373 (70.2) Normal delivery 3328 (62.6) 711 (61.7) 1138 (62.1) 234 (61.7) 1245 (63.7) Vacuum extraction 343 (6.4) 84 (7.3) 133 (7.3) 20 (5.3) 106 (5.4) Forceps extraction 56 (1.1) 12 (1.0) 21 (1.1) 1 (0.3) 22 (1.1) Neonatal birth weight (g), mean (SD) 3093.1 (457.7) 3101.5 (471.1) 3084.3 (449.7) 3073.1 (472.7) 3100.3 (454.3) 0.52a aOne-way analysis of variance. bw2 test. cComparison is between cesarean and vaginal routes.

Table 3 Adverse pregnancy outcomes of the four groups of women according to their blood phenotypes

Outcome Total Blood group (n ¼ 5320) A(n ¼ 1153) B (n ¼ 1832) AB (n ¼ 379) O (n ¼ 1956)

n (%) Crude/adjusted n (%) Crude/adjusted n (%) Crude/adjusted n (%) Crude/adjusted RR (95% CI)a RR (95% CI)a RR (95% CI)a RR (95% CI)a

Preeclampsia 350 (6.6) 100 (8.7) 1.7 (1.3–2.2) 113 (6.2) 1.2 (0.9–1.5) 32 (8.4) 1.6 (1.1–2.5) 105 (5.4) F 1.7 (1.3–2.3) 1.1 (0.8–1.4) 1.7 (1.1–2.6) (Reference) GDM 333 (6.3) 80 (6.9) 1.2 (0.9–1.7) 119 (6.5) 1.2 (0.9–1.5) 23 (6.1) 1.1 (0.7–1.7) 111 (5.7) F 1.2 (0.9–1.6) 1.1 (0.9–1.5) 1.1 (0.7–1.8) (Reference) Preterm delivery 543 (10.2) 125 (10.8) 1.2 (0.9–1.5) 194 (10.6) 1.1 (0.9–1.4) 39 (10.3) 1.1 (0.8–1.6) 185 (9.5) F 1.2 (0.9–1.5) 1.1 (0.9–1.4) 1.0 (0.7–1.5) (Reference) LBW 394 (7.4) 79 (6.9) 0.9 (0.7–1.2) 138 (7.5) 1.0 (0.8–1.3) 34 (9.0) 1.2 (0.8–1.8) 143 (7.3) F 0.9 (0.7–1.2) 1.0 (0.8–1.3) 1.1 (0.8–1.7) (Reference) SGA 178 (3.3) 35 (3.0) 0.8 (0.5–1.3) 58 (3.2) 0.9 (0.6–1.2) 14 (3.7) 1.0 (0.6–1.8) 71 (3.6) F 0.8 (0.5–1.2) 0.8 (0.6–1.2) 0.9 (0.5–1.7) (Reference)

Abbreviations: CI, confidence interval; GDM, gestational diabetes mellitus; LBW, low birth weight; RR, relative risk; SGA, small for gestational age. aAdjusted for age, parity, body mass index and other adverse pregnancy outcomes in the Table. of GA at delivery, route of delivery or neonatal birth weight among Upon further analysis, we found that the degree of RRs the four groups of women with different blood types. increased as the clinical manifestations of hypertensive disorders Table 3 compares the adverse pregnancy outcomes or pregnancy became more severe. The adjusted RRs of gestational hypertension, complications among women with A, B, AB, and O phenotypes. By mild preeclampsia and severe preeclampsia in A blood type were univariable analysis, women with A or AB blood types, but not B, 1.0 (95% CI, 0.6 to 1.8; P>0.05), 1.6 (95% CI, 1.1 to 2.3; had significantly higher risk of preeclampsia than O type P ¼ 0.01) and 2.1 (95% CI, 1.3 to 3.3; P<0.01), respectively, individuals. We used multivariable analysis to adjust for potential whereas the respective adjusted RRs in AB blood type were 1.1 (95% confounders for preeclampsia, including age, parity, body mass CI, 0.4 to 2.6; P>0.05), 1.6 (95% CI, 1.1 to 2.8; P ¼ 0.04) and 1.8 index as well as the other variables in the Table. Both A and AB (95% CI, 1.1 to 3.6; P ¼ 0.03). phenotypes were identified as independent risk factors for preeclampsia (RR, 1.7; 95% CI, 1.3 to 2.3; P ¼ 0.001 and RR, 1.7; 95% CI, 1.1 to 2.6; P ¼ 0.01, respectively). On the other hand, uni- Discussion and multivariable analyses did not show significant relationships The ABO blood group is the most important blood type system in between blood types and GDM, preterm delivery, LBW or SGA. humans especially in medicine. The distribution of ABO phenotypes

Journal of Perinatology ABO blood group and pregnancy outcomes C Phaloprakarn and S Tangjitgamol 110 varies across the globe, depending on racial/ethnic origins and It is well recognized that SGA shares the same underlying geographic regions. For example, A blood type is more common pathogenesis with preeclampsia. However, our study could not than the other blood types in Japan and European countries, demonstrate association of a particular ABO phenotype with the whereas O type predominates in the United States.20 O blood type is risk of having an SGA infant as we found with preeclampsia. This also the most common blood type found in Thai population might be due to the small number of subjects studied. Nevertheless, followed by B, A and AB types: 37.8% for O type, 34.8% for B, 20.5% we found that A blood type was a significant risk factor for for A and 6.9% for AB.21 Our study that focused only on pregnant coincidental events of SGA and preeclampsia (rather than SGA women also found similar distribution of blood types as those alone) with an adjusted RR of 3.4 (95% CI, 1.1 to 9.9; P ¼ 0.03). encountered in the general Thai population.21 Further studies with larger sample sizes might verify the potential Several studies have shown associations between particular ABO influence of ABO phenotype on this pregnancy outcome. phenotypes and pregnancy complications. In a study conducted by One previous study identified a higher prevalence of O blood May, British women with A blood type had a 2.7-fold risk of type in gravidas with GDM compared with non-GDM group (52.2% preeclampsia compared with O type individuals.13 Similarly, vs 33.3%).16 However, evaluation of the risk factors for GDM was Spinillo et al.14 and Hiltunen et al.12 found an increased risk of not the main purpose of such a study, so information on the RR of preeclampsia by 2.1- to 3.1-folds in Italian and Finnish gravidas this pregnancy complication in relation to maternal blood group with AB blood type compared with O type women. However, the was not reported. In our study, we found that the RRs of GDM impact of blood types on pregnancy complications was not observed among the four blood phenotypes were comparable. Similarly, we by Clark et al. who conducted a study on Scottish women.15 In our observed no associations of blood types with preterm delivery or study, we also found that both A and AB blood types were LBW. These negative results may serve as basic data for future significantly associated with preeclampsia; however, the 1.7-fold research to look for other genetic variants that may link to such risk in either blood group was smaller than those observed in complications. previous reports.12–14 Different results regarding the magnitude of This is the first study to evaluate the impact of ABO blood types associations among these studies might lie on various sample sizes, on maternal and neonatal outcomes in Asian population. The racial/ethnic origins studied, and population characteristics (that strength of our study was that it was conducted on a large cohort of is, primigravidas in the studies of May and Spinillo et al.vsa pregnant women. Additionally, all of the study population combination of primi- and multigravidas in the studies of Hiltunen underwent blood group testing, therefore information on blood et al., Clark et al. and our study). phenotypes was considered accurate. At the same time, some We also found a direct association of degree of RRs with severity drawbacks of this study were recognized. First of all, as this was a of preeclampsia in A and AB blood types. Our results were consistent retrospective study, data on other potential risk factors for the with the findings of Hiltunen et al.12 who reported RRs of 2.1 for outcomes being investigated were not available, such as, history of preeclampsia as a whole and 2.3 specifically for severe preeclampsia preeclampsia or GDM in prior pregnancies and so on. However, we in AB type individuals. Knowing the risk of specific type of adjusted for known potential confounders by using multivariable hypertensive disorders (that is, mild or severe preeclampsia) may be analysis. Second, the possible impact of minor red blood cell clinically useful because the maternal and neonatal prognosis as antibodies on adverse pregnancy outcomes could not be addressed well as pregnancy management depend on the severity of the disease. because the majority of the study population was not cross- There are several hypotheses for the observed relationship matched. Third, we did not include Rh-negative blood group between ABO blood types and preeclampsia. One possible subjects because of its low incidence (<0.5%) in Thai mechanism is due to an effect of von Willebrand factor, which was population.30 Consequently, impact on women with this particular found higher in non-O (A, B, and AB) compared with O type blood type was not determined. Lastly, the possible influences of individuals.22 Evidence suggests that von Willebrand factor can neonatal or paternal ABO blood types on pregnancy outcomes promote platelet aggregation/adhesion and atherosis formation could not be assessed because additional blood testing was not leading to endothelial dysfunction, which is known to be involved possible in our retrospective study. These limitations may be in the pathogenesis of preeclampsia.23–25 Other mechanisms may minimized in future and prospective studies. also get involved because our study found only an increased risk of In conclusion, our results demonstrated that maternal ABO preeclampsia in women with A and AB blood types, but not B type blood group was associated with the risk of preeclampsia, but not gravidas. Some inflammatory markers, that is, tumor necrosis with GDM, preterm delivery, LBW or SGA. A and AB blood types factor-alpha and soluble intercellular adhesion molecule 1 were were independent risk factors for preeclampsia and the degree of identified to be upregulated by single-nucleotide polymorphism RRs varied directly with the severity of the disease. Our findings rs651007 at the ABO locus, especially with the A allele.10,11,26 might have a role in identifying women at risk and to alert a As these markers are associated with preeclampsia,27–29 this is clinician to the possibility of a case of preeclampsia and proactive therefore another possible mechanism. early detection. At all times, we were aware of the limitations

Journal of Perinatology ABO blood group and pregnancy outcomes C Phaloprakarn and S Tangjitgamol 111 regarding the small proportion of A and AB phenotypes in our 15 Clark P, Walker ID, Govan L, Wu O, Greer IA. The GOAL study: a prospective population and of the modest increased risk of preeclampsia in examination of the impact of factor V Leiden and ABO(H) blood groups on either blood type. Hence, one could not draw a final conclusion haemorrhagic and thrombotic pregnancy outcomes. Br J Haematol 2008; 140: from our results until further research with various population 236–240. 16 Donma MM. Macrosomia, top of the iceberg: the charm of underlying factors. Pediatr groups further validate our findings. Int 2011; 53: 78–84. 17 American College of Obstetricians and Gynecologists Committee on Practice BulletinsFObstetrics. ACOG practice bulletin: diagnosis and management of Conflict of interest preeclampsia and eclampsia: number 33, January 2002. Obstet Gynecol 2002; 99: The authors declare no conflict of interest. 159–167. 18 American Diabetes Association. Gestational diabetes mellitus. Diabetes Care 2004; 27(Suppl 1): S88–S90. 19 Thaithamyanont P, Bhongvej S, Chittinond S. Intrauterine growth in Thai population. References J Pediatr Soc Thai 1984; 23: 99–105. 1 Storry JR, Olsson ML. The ABO blood group system revisited: a review and update. 20 Racial & ethnic distribution of ABO blood types. Available at: http://www.bloodbook. Immunohematology 2009; 25: 48–59. com/world-abo.html, Retrieved 9 February 2012. 2 Storry JR. Human blood groups: inheritance and importance in transfusion medicine. 21 Wiwanitkit V. ABO blood group of the Thais in Bangkok. Songkla Med J 2001; 19: J Infus Nurs 2003; 26: 367–372. 9–12. 3 Reid ME, Mohandas N. Red blood cell blood group antigens: structure and function. 22 Jenkins PV, O’Donnell JS. ABO blood group determines plasma von Willebrand factor Semin Hematol 2004; 41: 93–117. levels: a biological function after all? Tranfusion 2006; 46: 1836–1844. 4 Yip SP. Single-tube multiplex PCR-SSCP analysis distinguishes 7 common ABO alleles 23 Nadar SK, Al Yemeni E, Blann AD, Lip GY. Thrombomodulin, von Willebrand factor and readily identifies new alleles. Blood 2000; 95: 1487–1492. and E-selectin as plasma markers of endothelial damage/dysfunction and activation in 5 Oriol R, Candelier JJ, Mollicone R. Molecular genetics of H. Vox Sang 2000; 78(Suppl pregnancy induced hypertension. Thromb Res 2004; 113: 123–128. 2): 105–108. 24 Parra-Cordero M, Bosco C, Gonza´lez J, Gutie´rrez R, Barja P, Rodrigo R. 6 Garratty G. Blood groups and disease: a historical perspective. Transfus Med Rev 2000; Immunohistochemical expression of von Willebrand factor in the preeclamptic 14: 291–301. placenta. J Mol Histol 2011; 42: 459–465. 7 Smith AW, Aathithan S, Power EG, Abdulla Y. 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Maternal serum levels of association study identifies protein quantitative trait loci (pQTLs). PLoS Genet 2008; 4: TNF-alpha and IL-6 long after delivery in preeclamptic and normotensive pregnant e1000072. women. Mediators Inflamm 2010; 2010: 908649. 11 Pare´ G, Chasman DI, Kellogg M, Zee RY, Rifai N, Badola S et al. Novel association of 28 Szarka A, Rigo´ Jr J, La´za´r L, Beko G, Molvarec A. Circulating cytokines, chemokines and ABO histo-blood group antigen with soluble ICAM-1: results of a genome-wide adhesion molecules in normal pregnancy and preeclampsia determined by multiplex association study of 6578 women. PLoS Genet 2008; 4: e1000118. suspension array. BMC Immunol 2010; 11: 59. 12 Hiltunen LM, Laivuori H, Rautanen A, Kaaja R, Kere J, Krusius T et al. Blood group AB 29 Chavarrıá ME, Lara-Gonza´lez L, Garcıá-Paleta Y, Vital-Reyes VS, Reyes A. 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Journal of Perinatology Journal of Perinatology (2013) 33,94–97 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Preterm severe preeclampsia in singleton and twin pregnancies

DE Henry, TF McElrath and NA Smith Department of Obstetrics and Gynecology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

singletons, or with different manifestations of disease. A better Objective: We aimed to evaluate rates of delivery and clinical manifestations understanding of early, severe disease in multiple gestations may of preterm severe preeclampsia in singleton and twin gestations. aid our ability to care for multifetal pregnancies, and improve our Study Design: This retrospective cohort study included 86 765 deliveries understanding of the pathophysiology of the disease. from 2000 to 2009, including 3244 twins. Rates of delivery for severe We hypothesized that severe preterm preeclampsia would be preeclampsia among infants born 24 to 31 þ 6, and 32 to 36 þ 6 weeks more common among twin gestations, and that clinical gestation were calculated, and diagnostic criteria were compared. presentation of the disease may differ by twinning status. We therefore, primarily, aimed to compare the rates of delivery for Result: Re-term severe preeclampsia was more common in twin preterm severe preeclampsia between singleton and twin gestations pregnancies (2.4% vs 0.4%, P<0.001, relative risk 5.70 (95% confidence in a large cohort, and secondarily, to compare the clinical interval 4.47 to 7.26)). This was also true for deliveries from 24 to 31 þ 6 manifestations of disease in these two groups. (0.8% vs 0.2%, P<0.001) and 32 to 36 þ 6 weeks (1.7% vs 0.3%, P<0.001). Diagnostic criteria and disease manifestation including hemolysis elevated liver enzymes low platelet count syndrome, abruption Methods and growth restriction were similar between groups. We conducted a retrospective cohort study at a single institution including all deliveries of singleton or twin gestations from 2000 to Conclusion: Twin pregnancies are significantly more likely than 2009. An institutional database containing demographic, prenatal singletons to be delivered preterm for severe preeclampsia. Diagnostic and intrapartum data was used to identify all women with criteria and disease manifestation were similar in singletons and twins, at diagnoses of preeclampsia or hypertension, and delivery between 24 all gestational ages. and 0/7 and 36 and 6/7 weeks gestation. For all women who Journal of Perinatology (2013) 33, 94–97; doi:10.1038/jp.2012.74; delivered before 37 weeks, and carried a diagnosis of hypertension published online 7 June 2012 or preeclampsia, electronic medical records were reviewed. A Keywords: risk; diagnostic criteria; multifetal gestation; prematurity diagnosis of severe preeclampsia was made based on clinician diagnosis and validated by chart review using American College of Obstetricians and Gynecologists criteria.1 This included blood Introduction pressure >160/110 on two instances at least 6 h apart, symptoms of severe headache, visual changes, epigastric pain, transaminase Preeclampsia is a heterogeneous syndrome thought to complicate 5 values greater than twice normal, platelets 100 000, >5 g of to 8% of pregnancies.1 At term, the disease is easily managed by proteinuria in 24 h, oliguria (<500 ml in 24 h), severe fetal growth delivery of the fetus, although preterm severe disease can present a restriction or pulmonary edema. Only women who met the above diagnostic and therapeutic challenge.2,3 It is well-established that criteria for severe preeclampsia were included in the study. Patient women with twin pregnancies are at increased risk of hypertensive data including age, gravidity, parity, other intrapartum diagnoses disorders in pregnancy,4–9 however, prior studies have attributed a and preeclampsia diagnostic criteria were collected. The presence of risk to the entirety of gestation, rather than assessing when rates of hemolysis elevated liver enzymes low platelet count syndrome, disease begin to increase. Thus it is unknown whether twin clinical diagnosis of placental abruption, eclampsia and fetal pregnancies are affected by preeclampsia earlier than are criteria such as intrauterine growth restriction (estimated fetal Correspondence: Dr DE Henry, Department of Obstetrics and Gynecology, Brigham and weight <10%), and absent or reversed umbilical artery Doppler Women’s Hospital, Harvard Medical School, 75 Francis St, Boston, MA 02115, USA. flow, and intrauterine fetal death were noted. Owing to E-mail: [email protected] heterogeneous prenatal records, it was not possible to determine all This work was previously presented at the 2011 Society for Maternal-Fetal Medicine Annual medical comorbities. Meeting. Received 7 November 2011; revised 19 April 2012; accepted 7 May 2012; published online 7 June Patient demographics and intrapartum diagnoses were 2012 compared between women with twin and singleton gestations, and Risk of preterm severe preeclampsia DE Henry et al 95 between those who delivered from 24 and 31 þ 6 weeks and those Table 1 Characteristics of women with preterm severe preeclampsia by twinning from 32 and 36 6/7 weeks. At our institution, severe preeclamptics status who qualify for expectant management are delivered at 34 weeks. Singleton (N ¼ 352) Twin (N ¼ 79) P-value By using a 32-week threshold, we aimed to capture those patients Demographics Mean (s.d.) Mean (s.d.) that did not qualify for expectant management owing to the severity of their disease. The rate of preterm delivery for severe Age 31.0 (6.4) 32.7 (6.1) 0.040 preeclampsia was calculated for all gestational age categories. AgeX35 116 (33) 29 (37) NS The proportion of preterm deliveries accounted for by severe Gravidity 2.3 (1.7) 1.9 (1.0) 0.012 Parity 0.7 (1.0) 0.3 (0.7) 0.003 preeclampsia was also calculated for twins and singletons at each Gestational age at delivery 32.0 (3.0) 32.5 (2.6) NS gestational age category. Finally, we compared the criteria by which patients met the diagnosis of severe preeclampsia. The data Ethnicity 2 were analyzed using STATA 4.0. w tests and student’s t-tests were Caucasian 162 (46) 50 (63) NS used for comparison, and Yates’ correction was applied for low African–American 87 (25) 7 (9) 0.015 frequencies. Logistic regression was used to identify factors Asian 49 (14) 6 (8) NS associated with twin gestation among women with severe Hispanic 16 (5) 5 (6) NS preeclampsia. Unknown/Other 38 (11) 11 (14) NS

Mode of delivery N (%) N (%) P-value Results Vaginal 101 (29) 18 (23) There were 86 765 births during our study period, including 83 310 Cesarean 248 (71) 61 (77) NS singletons, 3244 twins and 211 higher-order multiples. There were 6259 preterm deliveries (4672 singletons, 1399 twins, and 188 Secondary diagnoses at delivery N (%) N (%) P-value higherorder multiples). Among those, 352 women with a singleton gestation and 79 women with a twin gestation delivered preterm Chronic hypertension 62 (18) 6 (8) 0.030 and had a diagnosis of severe preeclampsia as ascertained by PPROM 7 (2) 2 (3) NS Chorioamnionitis 0 0 NA diagnosis at delivery, and validated by chart review utilizing Preterm labor 7 (2) 8 (10) <0.001 American College of Obstetricians and Gynecologists criteria for History of prior preterm birth 44 (13) 4 (5) NS diagnosis of severe preeclampsia. The characteristics of women IUFD 3 (1) 0 NS with singleton and twin gestations were compared (Table 1). Women with a twin gestation were older (mean age 32.7 vs Abbreviations: NS, not significant; s.d., standard deviation. 31.0 years, P ¼ 0.04) than those with a singleton, and had fewer pregnancies and deliveries. 75% of women with a twin gestation Next, we evaluated these same characteristics within gestational were nulliparous compared with 55% of women with a singleton, age categories 24 to 31 6/7 and 32 to 36 6/7 weeks. Among women P<0.001. Gestational age at delivery was the same between groups. who delivered between 32 0/7 and 36 6/7 weeks gestation, those African–American women were more likely to have a singleton with twin gestations were significantly older and had fewer gestation than a twin gestation (P ¼ 0.015). The majority of pregnancies and deliveries. Those with twins were more likely to patients were delivered via cesarean section, although there was no have a diagnosis of preterm labor; 16% vs 2%, P<0.001, among difference in the cesarean delivery rate between singleton and twin women delivered between 24 and 31 6/7 weeks, and 7% vs 2%, gestations. Women with singleton gestations were more likely than P ¼ 0.066 among women delivered between 32 and 36 6/7 weeks. women with twin gestations to have a preexisting diagnosis of This difference was only statistically significant in the women who chronic hypertension (18% vs 8%, P ¼ 0.03). Women with twins delivered in the earliest gestational age category. were more likely to have a secondary diagnosis of preterm labor Rates of preterm delivery for severe preeclampsia were calculated (10% vs 2%, P<0.001). and compared (Figure 1). Over the entire population of births, Logistic regression was used to evaluate factors associated with women with twins were significantly more likely than those with a twining status among the cohort of women with a diagnosis of singleton to have a preterm delivery for severe preeclampsia (2.4% severe preeclampsia. Potential confounders were evaluated vs 0.4%, P<0.001), with a relative risk of 5.70 (95% confidence including age, race and chronic hypertension. In a model interval 4.47 to 7.26). This was true for women delivering between controlling for chronic hypertension and ethnicity, only race was a 24 0/7 and 31 6/7 weeks (0.8% vs 0.2%, P<0.001) and 32 to 36 6/7 significant predictor of twinning status, with African–American weeks (1.7% vs 0.3%, P<0.001). women less likely to have a twin gestation (odds ratio 0.28, 95% We evaluated the contribution of severe preeclampsia to preterm confidence interval (0.122 to 0.650)). birth rates. Among 1905 babies born between 24 0/7 to 31 6/7

Journal of Perinatology Risk of preterm severe preeclampsia DE Henry et al 96 weeks gestation, delivery for severe preeclampsia was equally pregnancies were significantly more likely than singletons common between singleton and twin gestations (9% vs 8%, to be delivered preterm due to severe preeclampsia. P ¼ 0.53). However, when evaluating those delivered between The clinical manifestations of preterm severe preeclampsia 32 0/7 and 36 6/7 weeks gestation, the rate of preterm delivery appeared to be similar between singleton and twins. Results for severe preeclampsia was greater for singletons than twins from this large, population-based cohort may be applicable to other (7% vs 5%, P ¼ 0.031). tertiary care centers, and may help further characterize a rare Finally, the diagnostic criteria for severe preeclampsia were disease process. compared. Rates of HELLP (hemolysis-elevated liver enzymes low The findings seen here are consistent with the large body of platelet count), abruption, eclampsia and fetal criteria were similar work showing increased hypertensive disorders among women with between singleton and twin gestations. Women with a twin multiple gestations.4–9 Sibai et al.8 reported the rates of severe gestation were more likely to be diagnosed with severe preeclampsia preeclampsia among twins as high as 6.4%. The rate of 2.4% here based on lab abnormalities alone (12% vs 3%, P ¼ 0.002). All is lower, but this is likely explained by the limitation of the current other categories of diagnostic criteria were similar between twins study to preterm disease, and by the larger cohort size. and singletons. Rates stratified by gestational age are reported Furthermore, the incidence of preterm severe preeclampsia did not in Table 2. differ by year of delivery within our cohort, supporting the internal validity of our estimates. Our rates of hemolysis elevated liver enzymes low platelet count syndrome and placental abruption are Discussion within the previously published estimates.8,10 Preterm severe preeclampsia carries high morbidity, and often Little literature exists describing the clinical manifestations presents a therapeutic dilemma for clinicians balancing the needs of preterm severe preeclampsia in either singleton or twin of mother and fetus. The risk of preterm severe preeclampsia has pregnancy. Although this study was underpowered todetect a not been well-defined, and it is unknown whether the difference in diagnostic criteria, there did not appear to be pathophysiology of preeclampsia in singleton and multiple significant differences between twin and singleton gestations, gestations differs. In this cohort of nearly 87 000 births, twin with the exception of increased deliveries for laboratory findings only in 32 to 36 þ 6 week twins. This may suggest that the underlying pathophysiology of preterm severe preeclampsia

2.50% * Singleton Twin is not altered in the setting of multifetal gestation. Data on N=79 how gestational age at onset and twinning status interact is 2.00% * * p < 0.001 limited, and our study suggests that although women with twins are more likely to develop early severe preeclampsia than women N=54 1.50% with a singleton gestation, the manifestations of this disease appears similar. 1.00% * This study has several important limitations. This was a N=25 retrospective investigation with the typical constraints on data 0.50% N=352 N=214 collection. As such, there are several potential confounders that

Rate of delivery for severe preeclampsia N=138 0.00% could not be accounted for, including maternal medical disease < 37 weeks 32-37 weeks 24-32 weeks (with the exception of chronic hypertension), mode of conception Figure 1 Rates of severe preeclampsia by twinning status and gestational age. and smoking status. However, the aim of this study was not to

Table 2 Severe preeclampsia diagnostic criteria by twinning status and gestational age at delivery

Diagnostic criteria 24 0/7–31 6/7 weeks P-value 32 0/7–36 6/7 weeks P-value

Singleton (N ¼ 138) Twins (N ¼ 25) Singleton (N ¼ 214) Twins (N ¼ 54)

Blood pressure alone 27 (20) 2 (12) NS 70 (33) 17 (31) NS HELLP 14 (10) 2 (8) NS 10 (5) 6 (11) NS Fetal growth restriction 23 (17) 4 (16) NS 27 (13) 3 (6) NS Abruption 4 (3) 1 (4) NS 8 (4) 1 (2) NS Serum laboratories 5 (4) 2 (8) NS 6 (3) 7 (13) 0.002

Abbreviations: HELLP, hemolysis elevated liver enzymes low platelet count; NS, not signiﬁcant. Values are represented as N (%).

Journal of Perinatology Risk of preterm severe preeclampsia DE Henry et al 97 assess risk factors for the development of the disease, but rather to Acknowledgments describe the incidence of a rare outcome among a large No financial support was provided for this project. population-based cohort. The contribution of maternal comorbidities to the risk of severe preeclampsia has been References established.11 Our goal was to compare rates and manifestations of 1 American College of Obstetricians and Gynecologists. Diagnosis and management of disease between singletons and twins. In our cohort, it is unlikely preeclampsia and eclampsia. ACOG Practice Bulletin No. 33. Obstet Gynecol 2002; 99: that chronic hypertension or race accounted for the increased risk 159–167. among women with twin gestations, as the incidence of chronic 2 Bombrys AE, Barton JR, Nowacki EA, Habli M, Pinder L, How H et al. Expectant hypertension and white race were actually greater among women management of severe preeclampsia at less than 27 weeks’ gestation: maternal and with a singleton. There were differences in age and parity perinatal outcomes according to gestational age by weeks at onset of expectant management. Am J Obstet Gynecol 2008; 199: 247.e1–247.e6. between twins and singletons that could not be controlled for, 3 Alanis MC, Robinson CJ, Hulsey TC, Ebeling M, Johnson DD. Early-onset severe however, these may be statistical more than clinical. For preeclampsia: induction of labor vs elective cesarean delivery and neonatal outcomes. example, the mean difference in age between groups was <2 years. Am J Obstet Gynecol 2008; 199: 262.e1–262.e6. Also, prior work has suggested that differences in parity do not 4 Long PA, Oats JN. Preeclampsia in twin pregnancy- Severity and pathogenesis. Aust N Z explain the increased rate of preeclampsia in twins.12 Finally, J Obstet Gynaecol 1987; 27:1–5. our study was limited to women who delivered between 24 0/7 and 5 Ros HS, Cnattingius S, Lipworth L. Comparison of risk factors for preeclampsia and gestational hypertension in a population-based cohort study. Am J Epidemiol 1998; 36 6/7 weeks gestation, therefore the results may not be 147: 1062–1070. applicable to women with severe preeclampsia before viability 6 Santema JG, Koppelaar I, Wallenburg HCS. Hypertensive disorders in twin pregnancy. or at term. Eur J Obstet Gynecol Reprod Biol 1995; 58: 9–13. Additional research is needed to investigate the relationship 7 Spellacy WN, Handler A, Ferre CD. A case-control study of 1253 twin pregnancies from between early severe preeclampsia and multifetal gestation. a 1982–1987 perinatal data base. Obstet Gynecol 1990; 75: 168–171. 8 Sibai BM, Hauth J, Caritis S, Lindheimer MD, MacPherson C, Klebanoff M et al. Prospective studies evaluating the effect of obesity, use of assisted Hypertensive disorders in twin versus singleton gestations. Am J Obstet Gynecol 2000; reproductive technology and other possible risk-modifiers of this 182: 938–942. relationship are needed. Our study provides a description of a rare 9 Barton JR, Sibai BM. Prediction and prevention of recurrent preeclampsia. Obstet but morbid disease and its relationship to twin gestations. These Gynecol 2008; 112: 359–372. findings may be useful in both antenatal surveillance as well as 10 Sibai BM, Barton JR. Expectant management of severe preeclampsia remote from term: patient counseling. patient selection, treatment, and delivery indications. Am J Obstet Gynecol 2007; 196: 514.e1–514.e9. 11 Catov JM, Ness RB, Kip KE, Olsen J. Risk of early or severe preeclampsia related to pre- Conflict of interest existing medical conditions. Int J Epidemiol 2007; 36: 412–419. 12 Hernandez-Diaz S, Toh S, Cnattingius S. Risk of pre-eclampsia in first and subsequent The authors declare no conflict of interest. pregnancies: prospective cohort study. BMJ 2009; 338: b2255.

Journal of Perinatology Journal of Perinatology (2013) 33, 112–118 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp ORIGINAL ARTICLE Fetal and postnatal brain MRI in premature infants with twin–twin transfusion syndrome

SL Merhar1, BM Kline-Fath2, J Meinzen-Derr3, KR Schibler1 and JL Leach2 1Perinatal Institute, Division of Neonatology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 2Department of Radiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA and 3Division of Epidemiology and Biostatistics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

becomes the ‘donor’ twin and develops oligohydramnios, and the Objective: To describe the findings on fetal and postnatal magnetic other becomes the ‘recipient’ twin and develops polyhydramnios resonance imaging (MRI) in premature infants with twin–twin transfusion and occasionally cardiac dysfunction. Both fetuses are at risk syndrome (TTTS) and to determine whether currently used staging systems for brain injury, intrauterine fetal demise and prematurity. The and other fetal and postnatal factors correlate with brain injury in this Quintero staging system, based on ultrasound and Doppler population. findings, has been used for several years and correlates with the 2 3 Study Design: We performed a prospective study of 22 premature risk of perinatal survival and later neurodevelopmental disability. infants with TTTS whose mothers had fetal MRIs. Postnatal brain MRI More recently, several groups have attempted to develop scoring was performed at term equivalent age (38 to 44 weeks) and medical systems incorporating cardiovascular features of the recipient 4,5 records were reviewed. Brain injury was scored on fetal and postnatal twin. There is controversy over whether these newer scoring 6 MRIs using an injury scale incorporating hemorrhagic and systems add any prognostic value to Quintero staging. nonhemorrhagic injury. Selective fetoscopic laser photocoagulation (SFLP) of vascular anastomoses has improved survival and outcomes in TTTS as Result: The median (range) gestational age (GA) was 31 weeks (26 to 35) compared with amnioreduction, but rates of neurological injury and and birth weight (BW) was 1296 g (762 to 2330). In all, 5/22 patients (23%) neurodevelopment impairment among survivors remain high.3,7–11 had brain injury seen on fetal MRI and 15/22 patients (68%) had brain Antenatal brain injury in TTTS is thought to be due to alterations injury seen on postnatal MRI. Quintero stage was the only predictor variable in cerebral blood flow, and postnatal brain injury is often due to that was significantly correlated with the total brain injury score (P ¼ 0.05). complications of prematurity. Prior studies of brain injury in TTTS Conclusion: Postnatal brain injury in premature infants with TTTS is have relied on postnatal ultrasound to determine the rates of correlated with Quintero stage. GA and BW are not predictive of brain neurological injury. However, premature infants can suffer injury in this cohort of infants. neurological injury during labor and delivery or in the first 24 h after Journal of Perinatology (2013) 33, 112–118; doi:10.1038/jp.2012.87; birth, so determining the timing of lesions using even early postnatal published online 28 June 2012 ultrasound may not be accurate. In addition, ultrasound is known to have low sensitivity and specificity to detect nonhemorrhagic brain Keywords: Quintero; Cincinnati; brain injury; neuroimaging injury in neonates, and does not predict later neurodevelopmental outcomes as well as magnetic resonance imaging (MRI).12–14 For thesereasons,MRImaybemoreusefulandaccuratetodetermine Introduction brain injury in both the fetal and early postnatal periods. Twin–twin transfusion syndrome (TTTS) is a serious complication The purpose of this study is to describe findings seen on fetal of twin gestation with an incidence of B10% of monochorionic and postnatal MRI in premature infants with TTTS and to pregnancies.1 TTTS occurs when unbalanced vascular anastomoses determine the correlation of various factors, including injury develop within the placenta, causing a discrepancy in the seen on fetal MRI and recipient cardiovascular status, with distribution of blood volume between the two fetuses. One twin neurological injury seen on MRI at term equivalent age.

Correspondence: Dr SL Merhar, Perinatal Institute, Division of Neonatology, Cincinnati Children’s Hospital, 3333 Burnet Ave ML 7009, Cincinnati, OH 45229, USA. Methods E-mail: [email protected] Received 14 March 2012; revised 18 May 2012; accepted 24 May 2012; published online 28 June All mothers who received a fetal MRI at the Fetal Care Center of 2012 Cincinnati for the evaluation of TTTS and who delivered at least Fetal and postnatal brain MRI in TTTS SL Merhar et al 113 one live twin at a Cincinnati area hospital from October 2009 to Table 1 Quintero staging with Cincinnati modification March 2011 were approached for participation in the study. The Stage Donor Recipient Recipient study was approved by the Institutional Review Boards from the cardiomyopathy three Cincinnati area hospitals from which the patients were recruited. Diagnosis of TTTS was based on sonographic criteria I Oligohydramnios Polyhydramnios as per standard practice: (1) the presence of monochorionic II Absent bladder Bladder seen diamniotic twins, (2) oligohydramnios in the donor twin with the III Abnormal Doppler Abnormal Doppler None largest vertical pocket <2 cm, (3) polyhydramnios in the recipient IIIa Mild twin with the largest vertical pocket >8 cm and (4) exclusion of an IIIb Moderate IIIc Severe alternative diagnosis. The pregnancies were prospectively staged 2 IV Hydrops Hydrops according to the Quintero staging system, along with the V Death Death Cincinnati modification of the Quintero stage.4 The Cincinnati modification incorporates the cardiomyopathy of the recipient twin and has been shown to correlate with recipient survival (Table 1). information: birth weight (BW), sex, race, Apgar scores, days of Fetal MRI was obtained as part of the standard practice on mechanical ventilation, surfactant, culture positive sepsis, proven all patients referred for TTTS and was performed on a 1.5T GE necrotizing enterocolitis, bronchopulmonary dysplasia and head Signa HDx (Waukesha, WI, USA) with a torso-phased array coil ultrasound results. without maternal/fetal sedation. Fetal imaging consisted of a single-shot fast spin echo T2-weighted image and gradient echo Statistical analysis T1-weighted images. T2 imaging of the fetal brain was performed Because of the small sample size and non-normal distribution of in axial, coronal and sagittal planes at a slice thickness of 3 to the data, nonparametric methods were used for all statistical tests 4 mm and field of view of 30 mm. Postnatal MRI was obtained at conducted. The Spearman’s correlation coefficient was used to term equivalent age (37 to 44 weeks) without sedation using the investigate the association between continuous predictors (for ‘feed and swaddle’ method.15 All postnatal scans were performed on example, Quintero stage, BW) and the continuous outcome a 1.5T GE Signa HDx using a standard neonatal brain protocol, variable of postnatal brain injury score (total, hemorrhagic and which consisted of the following sequences: sagittal 3D fast-spoiled nonhemorrhagic). The association between potential continuous gradient-recalled, axial fast-spin echo proton density/T2, axial predictor variables and the dichotomous outcome of brain injury multiplanar gradient-recalled T2* gradient-recalled echo and 15 versus no brain injury seen on postnatal MRI was tested using direction axial diffusion tensor imaging with average diffusion Wilcoxon Rank Sum test (nonparametric equivalent of the t-test). coefficient and trace maps. Statistical analysis was performed using SAS (Version 9.2, SAS Institute, Cary, NC, USA). Scoring of MRIs Fetal MRIs were evaluated by a single radiologist experienced in fetal MRI. Postnatal MRIs were evaluated by two independent Results radiologists blinded to the gestational age (GA) and clinical course A total of 22 patients were enrolled in the study from October 2009 of the infants. A scoring system incorporating white-matter injury, to March 2011. Patient demographics are shown in Table 3. There gray-matter injury and hemorrhagic injury was used for scoring was no difference in demographic and clinical characteristics both fetal and postnatal MRIs (Table 2). The system was modified between the study patients and those in which consent was not from the Woodward et al.12 scoring system, which has been shown obtained for study participation (n ¼ 9). See Supplementary to correlate with neurodevelopment outcome at 2 years of age. Table 6 for more detailed information on the 22 enrolled subjects. Injury was classified as hemorrhagic (including periventricular, Five subjects (23%) demonstrated brain injury on fetal MRI. intraventricular and cerebellar hemorrhage) or nonhemorrhagic Three fetuses had unilateral germinal matrix or intraventricular (including white- and gray-matter injury and malformations). hemorrhages, one had mild unilateral ventricular enlargement, one had enlarged extra-axial spaces and one had borderline Chart review ventriculomegaly with increased extra-axial fluid. In addition, After the infants were enrolled in the study, the prenatal chart was three fetuses had prominent cerebral venous sinuses, which was reviewed for the following information: Quintero stage at the time not considered injury. of presentation, Cincinnati modification at the time of 15/22 patients had some injury seen on postnatal MRI (injury presentation, donor versus recipient status, type of treatment, GA at score >0) (Figure 1). In all, 12 of these 15 patients had an injury treatment, intrauterine fetal demise, GA at delivery, and antenatal score >1. Injury scores ranged from 0 to 16 (of a maximum steroids. The infant’s postnatal chart was reviewed for the following possible 32). The types of injuries seen are shown in Table 4.

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Table 2 Scoring system for fetal and postnatal brain injury in TTTS Table 2 Continued

Nonhemorrhagic injury Cerebellar hemorrhage (total number) White matter None: 0 White-matter injury nature and extent 1–4: 1 Normal: 0 X5 or any larger than 1 cm: 2 Focal T1 or T2 signal foci (p2/hemisphere): 1 Intraventricular >2/hemisphere: 2 None: 0 Periventricular white-matter loss Unilateral: 1 None: 0 Bilateral: 2 Mild reduction (mild increase in ventricular size): 1 Abbreviation: TTTS, twin–twin transfusion syndrome. Marked reduction (associated significant increase in intraventricular size): 2 Cystic abnormalities Table 3 Demographic and clinical variables None: 0 Single focus <2 mm: 1 Characteristic (n ¼ 22) Median Median Range Multiple or >2 mm: 2 (donors) (recipients) Ventricular dilatation Normal: 0 Gestational age, weeks 31 31 26–35 Moderate: 1 Birth weight, g 1292 1300 762–2330 Marked: 2 Quintero stage III III I–IV Abnormality of corpus callosum Cincinnati stage IIIc IIIc I–IV None: 0 Age at fetal MRI, weeks 22 22 17–26 Focal thinning: 1 Age at postnatal MRI, weeks 40 40 39–42 Global thinning: 2 Anomaly of corpus callosum n ¼ 11 n ¼ 11 None: 0 Male 6 7 Dysgenesis: 1 White 10 10 Absence: 2 IUFD of one twin 1 1 Gray matter Treated with SFLP 9 9 Cortical injury (nonmalformative) Received antenatal steroids 11 11 None: 0 Required mechanical ventilation 5 2 Single: 1 NEC 0 0 Multiple: 2 Sepsis 0 0 Gyral maturation BPD 3 1 Normal for term: 0 2–4 week delay (mild simplification): 1 Abbreviations: BPD, bronchopulmonary dysplasia; IUFD, intrauterine fetal demise; MRI, Significant delay >4 weeks: 2 magnetic resonance imaging; NEC, necrotizing enterocolitis; SFLP, selective fetoscopic laser photocoagulation. Gyral malformation Heterotopia: 1 Transmantle or schizencephaly: 2 Several infants had more than one finding on postnatal MRI. All Diffuse malformation, unilateral: 3 Diffuse malformation, bilateral: 4 the five patients with injury seen on fetal MRI had postnatal injury. Size of subarachnoid space The prominent cerebral venous sinuses seen on fetal MRI had all Small/normal: 0 been resolved on postnatal MRI. Only 4 of the 15 patients with Mild enlargement: 1 brain injury seen on MRI had any abnormalities seen on head Moderate global enlargement: 2 ultrasound, although two patients had no head ultrasounds performed during their neonatal intensive care unit stay. Head Hemorrhagic injury ultrasounds were obtained by the clinical team and were not part Periventricular (score each side) of the study protocol. None: 0 Results of the statistical analysis indicated that Quintero stage <25% of ventricular surface: 1 was the only predictor variable that was significantly correlated 25–50% of ventricular surface: 2 with the total brain injury score (Spearman’s correlation ¼ 0.43, >50% of ventricular surface: 3 P ¼ 0.05). Cincinnati stage, GA, injury on fetal MRI, Apgar scores, donor versus recipient status, sex, intrauterine fetal demise of twin

Journal of Perinatology Fetal and postnatal brain MRI in TTTS SL Merhar et al 115

Figure 1 Representative fetal and postnatal magnetic resonance imaging (MRI) of recipient twin with twin–twin transfusion syndrome (TTTS), Quintero and Cincinnati stage IV.

Table 4 Postnatal brain injury in 22 infants with TTTS brain injury score and all other potential predictor variables in Postnatal brain injury Donor Recipient Total Total donors and recipients separately, and found that BW was the only (n ¼ 11) (n ¼ 11) (n ¼ 22) % factor signiﬁcantly correlated with hemorrhagic injury score in recipients (Spearman’s correlation ¼ 0.65, P ¼ 0.02). Intraventricular/periventricular 4 3 7 32% hemorrhage White-matter volume loss 4 3 7 32% Discussion Ventriculomegaly The main objectives of our study were to describe the patterns of Unilateral 1 1 2 9% fetal and postnatal brain injury as seen on MRI in a cohort of Bilateral 2 1 3 14% Cerebral volume loss 3 2 5 23% premature infants with TTTS and to determine clinical predictors Periventricular white-matter injury 2 2 4 18% of postnatal brain injury. Multiple prior studies have looked at Migrational anomalies (cleft with gray- 1015%prenatal brain injury in infants with TTTS. Most of these studies matter heterotopia) exclusively looked at postnatal ultrasound in the ﬁrst 24 to 48 h of Cerebellar injury 1 1 2 9% life to describe prenatal brain injury, assuming that lesions seen

Abbreviation: TTTS, twin–twin transfusion syndrome. this early were acquired prenatally. The incidence of brain injury seen on head ultrasound in these studies ranged widely from 6 to 64%, and definitions of brain injury varied.16–23 We saw some and BPD were not significantly associated with the brain injury evidence of brain injury on postnatal MRI in 15/22 (68%) infants score. Although not statistically significant, higher total brain in our study, and 12/22 (54%) had injury scores >1 using our injury score appeared to trend with higher GA at treatment scale. Only 4 of the 15 infants had any evidence of brain injury on (Spearman’s correlation ¼ 0.34, P ¼ 0.13), birth weight head ultrasound, which confirms prior studies that MRI is more (Spearman’s correlation ¼ 0.32, P ¼ 0.14) and days of mechanical sensitive and specific than ultrasound,12,13 especially for white- ventilation (Spearman’s correlation ¼ 0.37, P ¼ 0.09). The matter injury. Cincinnati score (Spearman’s correlation ¼ 0.48, P ¼ 0.02) and Only one study has specifically looked at fetal MRI in TTTS.24 the Quintero score (Spearman’s correlation ¼ 0.54, P ¼ 0.009) In this study, 12/24 of the donor twins had enlarged cerebral were both significantly correlated with the hemorrhagic injury venous sinuses on fetal MRI, and 2/24 had MRI findings of score. Days of mechanical ventilation were significantly correlated cerebral malformations (one with alobar holoprosencephaly and with the nonhemorrhagic injury score (Spearman’s correlation ¼ one with a frontal lobe cortical malformation). In the recipient 0.49, P ¼ 0.02). See Supplementary Table 7 for all Spearman’s twins, cerebral ischemia or IVH that was not demonstrated correlations. on prenatal ultrasound was noted on the fetal MRI images Given that the Cincinnati modification describes the in 2/24, and cerebral venous sinus enlargement was seen in cardiovascular disease of the recipient but not the donor, we did an 3/24. We only rarely saw cerebral venous sinus enlargement on the analysis to evaluate the donor and recipient twins separately. We fetal MRIs (in two donors and one recipient), and did not found that the brain injury scores of donors and recipients were see this finding on the postnatal MRIs. This finding most likely correlated differently with the Cincinnati and Quintero staging reflects venous hypertension in the cerebral circulation. Other (Table 5). We also looked at the relationship between the postnatal findings on fetal MRI in our cohort were as follows; enlarged

Journal of Perinatology Fetal and postnatal brain MRI in TTTS SL Merhar et al 116

Table 5 Pearson’s correlation of staging of TTTS and postnatal brain injury infants with TTTS. It is estimated that 2 to 20% of very low BW score infants suffer cerebellar hemorrhage,32–35 which is better seen on 34,35 Quintero Cincinnati MRI than on ultrasound. Cerebellar injury has been related to 34,36 stage modification poor neurodevelopment outcomes in former preterm infants. The mechanism of injury is thought to be similar to the Recipients pathogenesis of IVH, so it is not surprising that 2/22 infants in our Total-injury score 0.37, P ¼ 0.25 0.39, P ¼ 0.23 study showed evidence of cerebellar injury on postnatal MRI. Hemorrhagic injury score 0.43, P ¼ 0.18 0.71, P ¼ 0.01 We evaluated two infants whose co-twins died after laser Nonhemorrhagic injury score 0.15, P 0.65 0.11, P 0.74 ¼ ¼ surgery. One infant, born at 35 weeks, had no brain injury. Donors The other, born at 32 weeks, had intraventricular hemorrhage Total-injury score 0.54, P ¼ 0.08 0.19, P ¼ 0.58 bilaterally, as well as bilateral cerebellar hemisphere ischemic Hemorrhagic injury score 0.72, P ¼ 0.01 0.23, P ¼ 0.50 injury. Multiple reports of brain injury after co-twin demise exist 8,27,37–40 Nonhemorrhagic injury score 0.12, P ¼ 0.72 0.10, P ¼ 0.77 in the literature. The surviving twin is thought to exsanguinate into the dead co-twin, leading to hypoxic brain Abbreviation: TTTS, twin–twin transfusion syndrome. Bold entries indicate statistically significant correlations, with p<0.05. injury in the survivor, which may manifest as polymicrogyria, hemorrhage, ventriculomegaly or delayed sulcation. The selective fetoscopic laser photocoagulation procedure should protect the surviving twin by eliminating the vascular anastomoses in the extra-axial spaces, unilateral ventricular dilation and placenta, but some anastomoses remain that may allow for intraventricular hemorrhage (grades I to II). As the fetal MRIs in transfer of blood volume into the low resistance circulation of the our cohort were obtained before laser treatment (at a median of 22 expired fetus. weeks gestation), they may not have demonstrated injury acquired In our statistical analysis, we found that the Quintero stage at between the MRI and birth. One patient had gray-matter presentation was the only statistically significant predictor for total heterotopia and polymicrogyria seen on postnatal MRI but had a brain injury score. The Quintero staging system was initially normal fetal MRI. Polymicrogyria is thought to occur during or developed to describe the severity of TTTS, and worsening Quintero after the neuronal migration period.25 It has been described before stage over time appears to be associated with poorer outcomes.41 in case reports of infants with TTTS,26–28 and in the infant in our When evaluating donors and recipients separately, we found that study it may have been due to a vascular insult that occurred after the Cincinnati modification better predicted hemorrhagic injury in the fetal MRI, performed at about 20 weeks in this patient. recipients while the Quintero score better predicted hemorrhagic Multiple types of brain injury have been described in infants injury in donors. Neither staging system was well-correlated with with TTTS. The most common types seen are germinal matrix/ the nonhemorrhagic injury score. It is unclear why hemorrhagic intraventricular hemorrhage and white-matter injury.16–18,23,29 injury was better correlated with the staging systems, but we Germinal matrix hemorrhage/IVH is due to bleeding from the hypothesize that the vascular changes in TTTS as both Quintero germinal matrix, a highly cellularized and vascular region present and Cincinnati stages increase may contribute more to germinal from B10 to 32 weeks of gestation.30 The hemodynamic matrix bleeding than to hypoxia-ischemia leading to white-matter disturbances in fetuses with TTTS leading to bleeding from the injury. The Cincinnati modification better captures the vascular thin-walled vessels within the germinal matrix could explain the changes of the recipient twin, while the disease state of the donor high incidence (7/22) seen in our cohort of patients. Only three of twin is better described by the Quintero stage. the seven patients who eventually developed germinal matrix The strengths of our study include its prospective nature, hemorrhage/IVH had evidence on fetal MRI, so some of the allowing for a predetermined protocol for the fetal and postnatal patients almost certainly developed this complication perinatally or MRI scans. However, only babies who survived to neonatal postnatally, and it may have been related to prematurity rather intensive care unit discharge were included, which may have than TTTS. White-matter injury, both cystic and diffuse, has also introduced selection bias. Given the timing of the fetal and frequently been described in infants with TTTS.16–18,23,29 The postnatal scans, it is difficult to truly know what brain injury was preterm brain responds to inflammation and ischemia by pre- antenatal (and thus TTTS related) and which injury was oligodendrocyte necrosis or apoptosis and reactive astrocytosis, postnatal (and therefore potentially prematurity related). In which are thought to lead to later myelination deficits.31 Also, the addition, we were unable to include a control group, although it disturbed blood flow in fetuses with TTTS could explain the white- is difficult to determine an appropriate control group for this matter injury, ranging from mild white-matter volume loss to cohort of infants. The small sample size limited our statistical periventricular leukomalacia, in 7 of the 22 babies in our study analysis, as we found some trends that were not significant at the cohort. Cerebellar hemorrhage has not been described previously in P ¼ 0.05 level.

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In conclusion, TTTS is associated with a high risk of brain of amniocentesis vs laser photocoagulation for the treatment of twin-to-twin injury in survivors, and the incidence and severity of injury is transfusion syndrome. Am J Obstet Gynecol 2010; 203(5): 444.e441–447. related to the Quintero stage. Neonatologists should be aware of the 12 Woodward LJ, Anderson PJ, Austin NC, Howard K, Inder TE. Neonatal MRI to high risk of brain injury and neurodevelopment disability in predict neurodevelopmental outcomes in preterm infants. N Engl J Med 2006; 355(7): 685–694. survivors of TTTS, regardless of GA at birth. 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