Effect of Delayed vs Early Umbilical Cord Clamping on Iron Status and Neurodevelopment at Age 12 Months: A Randomized Clinical Trial

Importance  Prevention of iron deficiency in infancy may promote neurodevelopment. Delayed cord clamping (DCC) can prevent iron deficiency during the first 6 months of life. However, no data are available on long-term effects on infant outcomes in relation to time for umbilical cord clamping.

Objective  To investigate effects of DCC, as compared with early cord clamping (ECC), on infant iron status and neurodevelopment at age 12 months in a European setting.

Design, Setting, and Participants  Randomized clinical trial of 382 full-term infants born after a low-risk pregnancy at a Swedish county hospital. Follow-up at 12 months included evaluation of iron status (ferritin level, transferrin saturation, transferrin receptor level, reticulocyte hemoglobin level, and mean cell volume) and parental assessment of neurodevelopment by the Ages and Stages Questionnaire, second edition (ASQ).

Interventions  Infants were randomized to DCC (≥180 seconds after delivery) or ECC (≤10 seconds after delivery).

Main Outcomes and Measures  The main outcome was iron status at age 12 months; the secondary outcome was ASQ score.

Results  In total, 347 of 382 infants (90.8%) were assessed. The DCC and ECC groups did not differ in iron status (mean ferritin level, 35.4 vs 33.6 ng/mL, respectively; P = .40) or neurodevelopment (mean ASQ total score, 229.6 vs 233.1, respectively; P = .42) at age 12 months. Predictors of ferritin levels were infant sex and ferritin in umbilical cord blood. Predictors of ASQ score were infant sex and breastfeeding within 1 hour after birth. For both outcomes, being a boy was associated with lower results. Interaction analysis showed that DCC was associated with an ASQ score 5 points higher among boys (mean [SD] score, 229 [43] for DCC vs 224 [39] for ECC) but 12 points lower among girls (mean [SD] score, 230 [39] for DCC vs 242 [36] for ECC), out of a maximum of 300 points (P = .04 for the interaction term).

Conclusions and Relevance  Delayed cord clamping did not affect iron status or neurodevelopment at age 12 months in a selected population of healthy term-born infants. However, it may not be possible to demonstrate minor effects on neurodevelopment with the size of the study population and the chosen method for assessment. The current data indicate that sex may influence the effects on infant development after DCC in different directions. The magnitude and biological reason for this finding remain to be investigated.

Trial Registration  clinicaltrials.gov Identifier: NCT01245296

Immediately after birth, the newborn infant still shares its blood circulation with the placenta and umbilical cord. As the arteries in the umbilical cord start to constrict, uterine contractions contribute with a net inflow of blood from the placenta into the newborn, totaling approximately 30% extra blood volume.¹ This transfusion will be impaired if immediate or early cord clamping (ECC) is performed, while a delay in clamping for 2 to 3 minutes will allow for a full transfusion. This placental transfusion has been shown to result in an increased hemoglobin (Hb) level and hematocrit in the neonatal period.² During the last decade, several studies have shown that the increased blood volume after delayed cord clamping (DCC) also contributes to higher ferritin levels at 4 to 6 months of life and possibly also helps to prevent infant iron deficiency (ID).^3,4

In animal models, ID has repeatedly been shown to affect dopamine metabolism, myelination, and energy metabolism.^5,6 In infancy, ID has also been associated with impaired neurodevelopment, including both gross motor function and fine motor development, and adverse behavior.⁷ Iron supplementation to populations with increased risk for ID improves psychomotor outcome in infancy and reduces behavioral problems.^8,9

We previously demonstrated in a randomized clinical trial that DCC, as compared with ECC, is associated with a higher neonatal Hb level and a lower rate of neonatal anemia as well as a higher ferritin level and less ID at age 4 months.¹⁰ No adverse effects of the intervention were found.^10,11 At age 4 months, the infants in the 2 groups had a comparable incidence of symptoms of infection and similar neurodevelopment.¹²

We hypothesized that DCC after delivery has long-lasting effects and that it improves iron status and neurodevelopment at age 12 months.

This study is a secondary analysis of a randomized clinical trial conducted at the Hospital of Halland, Halmstad, Sweden. The trial was approved by the Regional Ethical Review Board of Lund University. In short, pregnant women were considered eligible if they were nonsmoking, healthy, and carrying a low-risk singleton pregnancy with an expected vaginal delivery at term, as previously described.¹⁰ They were informed about the study at antenatal care units in the area around Halmstad. Quiz Ref IDAfter written informed parental consent was obtained and when delivery was imminent, 400 newborn infants were randomized to DCC at 180 seconds or more after delivery or to ECC before or at 10 seconds after birth, as previously described (Figure).¹⁰ Neither the mother giving birth nor the midwife performing the intervention could be blinded, but all staff involved in collection or analyses of blood samples or administration of the Ages and Stages Questionnaire, second edition (ASQ) were blinded to the allocation group. Early cord clamping was the standard method at the hospital during the inclusion period between April 16, 2008, and May 22, 2009. Quiz Ref IDInfants were scheduled for follow-up visits at ages 4 and 12 months.

The primary outcome of this randomized clinical trial was the ferritin level at age 4 months, as previously reported.¹⁰ For the 12-month follow-up, the following outcomes were prespecified: Hb level, iron status (as assessed by ferritin level, transferrin saturation [TS], soluble transferrin receptor [sTfR] level, and mean cell volume [MCV]), and neurodevelopment (as assessed by the ASQ [ASQ total score and scores on 5 subdomains: communication, gross motor, fine motor, problem solving, and personal-social]). As a post hoc outcome, we also analyzed determinants present at birth or age 4 months for ferritin and ASQ score at age 12 months.

The follow-up visit included blood sampling, weight measurement, and length measurement. Venous blood sampling was performed after application of a local anesthetic (lidocaine, 2.5%, and prilocaine, 2.5%). One month before the planned 12-month visit, parents received a letter with the ASQ and a 3-day food diary.

Blood samples were analyzed for the following: complete blood cell count (Hb level, hematocrit, and MCV), iron status (serum ferritin level, TS, and sTfR level), and C-reactive protein (CRP) level. Blood samples for complete blood cell counts were collected in ethylenediaminetetraacetic acid (EDTA) tubes (BD Vacutainer) and analyzed using the automated hematology analyzer Sysmex XE 2100 (Sysmex). Iron status indicators and CRP were collected in serum separator tubes (BD Vacutainer) and analyzed using a Cobas 6000 analyzer (Roche Diagnostics).

Anemia was defined as an Hb level lower than 11.0 g/dL (to convert to grams per liter, multiply by 10.0). Low MCV, ferritin level, and TS were defined as MCV lower than 70 fL, ferritin level lower than 12 ng/mL (to convert to picomoles per liter, multiply by 2.247), and TS less than 10%, respectively.¹³ We defined the upper reference limit for sTfR (5.9 mg/L) by the mean value plus 2 SDs of the values from the 330 study participants with no anemia and normal MCV, ferritin level, and TS. Iron deficiency was defined as at least 2 of 4 iron store indicators (ferritin level, TS, sTfR level, MCV) outside reference values. Iron deficiency anemia was defined as anemia in combination with ID.¹⁴ As inflammation is known to influence iron status markers,¹⁵ blood samples with a CRP concentration of 10 mg/L or greater (to convert to nanomoles per liter, multiply by 9.524) were excluded from analysis.

The ASQ¹⁶ was used to assess parent-reported infant development. Permission was obtained from Paul H. Brookes Publishing Co to translate the ASQ 12-month questionnaire to Swedish for use in the study. We created cutoff scores according to the ASQ manual, ie, by subtracting the mean score with 2 SDs. If the ASQs were not completely answered, scores were adjusted according to the ASQ manual.

Parents were asked to record, in detail, all food and beverages the child was given during 3 days, in weight (grams) or volume (milliliters). The diary and instructions were included in the letter sent 1 month before the 4- and 12-month visits.

The sample size for the primary outcome at 4 months was estimated to find a difference of 29% in geometric mean serum ferritin level between the 2 randomization groups with a power of 80% and a significance level of P < .05. A post hoc analysis of the 12-month data showed that a difference between the 2 groups of 7.5% in geometric mean ferritin level and a 12-point difference in the ASQ total score were needed to reach statistical significance (with a type I error rate of .05 and power of 80%).

Data were analyzed both according to intention to treat and per protocol. For comparisons, t test, Mann-Whitney U test, and Fisher exact test were used as appropriate. For 95% confidence interval across groups, the Hodges-Lehmann estimator was used. Ferritin was log₁₀ transformed for analysis. We used SPSS for Windows, version 18.0 (SPSS Inc). Relative risks and 95% confidence intervals were calculated by the web-based JavaStat calculator.¹⁷

Calculations of Spearman rank correlation coefficient (ρ) were used to determine neonatal and 4-month variables that were associated with outcomes at 12 months (ferritin level and ASQ total score). Variables with a correlation significance of P < .05 for each outcome were then entered into a linear regression model and analyzed by stepwise backward selection, leaving only variables with P < .05 in the model.

At 12 months, 347 of 382 infants (90.8%) returned for blood sampling and/or were assessed by the ASQ. Blood samples were collected from 337 of 382 infants (88.2%), of whom 174 of 193 (90.2%) belonged to the DCC group and 163 of 189 (86.2%) to the ECC group (P = .27) at a mean (SD) age of 366 (8) days in the DCC group and 366 (7) days in the ECC group (P = .92) (Figure). Baseline and background characteristics are presented in Table 1. Altogether, ASQ data were obtained for 340 infants (89.0%) with a completed ASQ, including 172 of 193 infants (89.1%) in the DCC group vs 168 of 189 infants (88.9%) in the ECC group (P > .99) (Figure). The date for answering the ASQ was given by 307 parents, showing that infants were a mean (SD) of 363 (9) days old when the assessments were done, with no difference between the groups (mean [SD] age, 362 [10] days for the DCC group vs 364 [8] days for the ECC group; P = .14). Data were excluded if blood sampling or the ASQ was done more than 1 calendar month before or after the infant reached age 12 months. For this reason, 1 blood sample in the DCC group was excluded, as were 3 sets of ASQ data (2 in the DCC group and 1 in the ECC group). A total of 32 blood samples with a CRP level of 10 mg/L or greater (9.5%) were excluded from analysis, including 11 (6.7%) in the ECC group and 21 (12.1%) in the DCC group (P = .10).

There were no differences between the 2 groups in Hb level or any of the iron status indicators at 12 months (Table 2). The proportions of cases outside reference ranges for anemia and iron store indicators did not differ between the groups (Table 2). The results did not change when data were analyzed per protocol.

Neurodevelopment at 12 months did not differ between the groups for the ASQ total score or 5 subdomain scores or for the proportion of infants below the cutoff level (Table 3). The results did not change when data were analyzed per protocol.

In univariate analyses, ferritin level at 12 months showed significant positive correlations with umbilical cord ferritin level and TS and negative correlations with umbilical cord Hb level and male sex. Multivariate analyses demonstrated that umbilical cord blood ferritin level and infant sex remained independent significant predictors for ferritin level at 12 months (Table 4). Variables at 4 months with significant correlations with 12-month ferritin level included iron status indicators (positive correlation) and body weight (negative correlation). Variables at 12 months that correlated with 12-month ferritin levels were body weight and nutritional intake (negative correlations) and the infant’s age at blood sampling (positive correlation) (Table 1).

The ASQ total score at 12 months was positively associated with female sex, Apgar score at 5 minutes, and breastfeeding within the first hour of life. None of these variables differed between the 2 randomization groups (Table 1). Infant age at testing, within the interval of 11 to 13 months, positively correlated with ASQ score. In multivariate linear regression analysis, infant sex, breastfeeding within the first hour of life, and postnatal age at testing remained significant predictors for ASQ score at 12 months (Table 4). In univariate analyses, the 12-month ASQ did not correlate with gestational age, birth weight, Hb level, or any of the iron status indicators in umbilical cord blood. The ASQ total score at 12 months correlated with the ASQ score at 4 months (ρ = 0.401; P < .001; n = 337), while breastfeeding at 4 months had no effect on the ASQ score at 12 months (Table 1).

Because infant sex correlated independently with both ferritin level and ASQ score at 12 months, sex and randomization group were entered as variables in an analysis of covariance model. No interaction was found in the model concerning the 12-month ferritin level, but in the ASQ model there was a significant interaction. Randomization to DCC compared with ECC was associated with a 12-point-lower ASQ score for girls (mean [SD] score, 230 [39] vs 242 [36] points, respectively) and a 5-point-higher score for boys (mean [SD] score, 229 [43] vs 224 [39] points, respectively) (P = .04 for the interaction term).

We previously demonstrated, in a randomized clinical trial, that DCC for 3 minutes or more increases neonatal Hb and IgG levels and improves iron stores at age 4 months without noticeable adverse effects.^10,12 In this study, we assessed the same cohort of term-born infants at age 12 months. We hypothesized that the improved iron status at age 4 months would prevent ID at age 12 months and also promote infant neurodevelopment. However, we were unable to demonstrate any differences in iron status and in neurodevelopment between the randomization groups at 12 months.

Quiz Ref IDDelayed cord clamping adds about 30% extra blood volume to the newborn. As the red blood cells undergo hemolysis, the hemoglobin is metabolized to enrich the infant’s iron stores; consequently, DCC increases iron stores at ages 4 to 6 months and also seems to prevent ID at age 4 months. Except for 1 small study (n = 28), published in 1941,¹⁸ that demonstrated a higher mean corpuscular Hb level in 8- to 10-month-old infants subjected to DCC, no study investigating effects of timing for umbilical cord clamping has, to our knowledge, a longer follow-up period than 6 months.

In this study, iron stores were assessed by different indicators of iron status: Hb level, MCV, reticulocyte Hb level, ferritin level, TS, and sTfR level. Even when comparing the levels of each of these indicators, we could not demonstrate any differences in iron status between the 2 randomization groups. We also examined the proportion of infants with ID in our population by using a combination of 4 iron status indicators (ferritin level, MCV, TS, and sTfR level), but no significant difference was found in the proportion of ID between the 2 groups. We found that ferritin level in umbilical cord blood was a strong predictor of ferritin level at age 12 months.

Iron deficiency is associated with serious complications such as ID anemia and suboptimal neurodevelopment,¹⁹ and iron substitution in infancy prevents ID anemia and improves neurodevelopment.⁸ As for ID anemia, we did only find 1 infant fulfilling the criteria for this condition at age 12 months, and although this infant belonged to the ECC group, this finding is not associated with a significant difference between groups. In this study, the prevalence of ID at age 12 months, defined as a serum ferritin level lower than 12 ng/mL, was much lower (1.7%) than in a previous Swedish study (26%) published in 1998.²⁰ A national recommendation that emphasized avoidance of cow’s milk as a beverage and promoted iron-fortified cereals during the second half of the first year may have led to this improvement in iron status.²¹ This is supported by 2 cohort studies in Iceland showing a similar decrease of infants with low ferritin levels (<12 ng/mL) from 41% to 5.8% between 1997 and 2005 after a change in recommendations in 2003.^22,23 To be able to measure possible effects of DCC and ECC on ID and ID anemia at age 12 months, we conclude that either a considerably larger study group is needed or the study should be performed in a part of the world where ID or ID anemia is more prevalent.

Neurodevelopment in relation to time of cord clamping has previously only been reported twice to our knowledge. Mercer et al²⁴ demonstrated in boys born very preterm that DCC had a protective effect against motor disability at age 7 months. The other study was the current cohort in which development was assessed by the ASQ at age 4 months, showing no differences in total scores but higher scores in the problem-solving domain and lower scores in the personal-social domain in the DCC group.¹² In the present study, neither the ASQ total score nor the subscores of the different domains differed between the groups. Furthermore, we did not find any associations between iron status at birth, iron status at age 4 months, or ASQ total score at age 12 months. However, interaction analysis showed that the effect of the intervention differed according to infant sex, as DCC seemed to lower ASQ scores in girls and increase them in boys, a finding that prompts a more detailed follow-up of these children. Infants who were breastfed during the first hour of life also had higher ASQ scores, and there was a relation between Apgar score and ASQ score. In the study population, early breastfeeding was associated with uncomplicated vaginal delivery and increased Apgar scores, which may explain the correlation between early breastfeeding and ASQ score at age 12 months. A limitation is that the ASQ is a screening tool designed to find children at risk for neurodevelopmental disability and as such may not be optimal for assessment of neurodevelopment in a cohort of mainly healthy infants. Still, the ASQ has been extensively validated and shown to have good specificity and sensitivity in detecting developmental problems, and we regard it as a useful tool that is easy to administer in a relatively large study population.

Quiz Ref IDA main limitation of this study is the sample size. It was calculated to demonstrate a difference in ferritin level at age 4 months, and as no earlier studies on cord clamping had studied iron status and neurodevelopment at age 12 months, calculations on sample size for these outcomes could not be performed before commencement of the study.

Quiz Ref IDAlthough DCC improved hematological status in the newborn period and iron status at age 4 months, it did not affect iron status or neurodevelopment assessed by the ASQ at age 12 months. Future research in this area should be directed at studying development at later ages and at assessing long-term effects of DCC on iron status and development in communities with higher rates of ID and anemia. The possible reverse effect on girls’ and boys’ development might also be taken into account.

Corresponding Author: Ola Andersson, MD, PhD, Department of Women’s and Children’s Health, Uppsala University, SE-751 85 Uppsala, Sweden (ola.andersson@kbh.uu.se).

Accepted for Publication: October 8, 2013.

Published Online: April 21, 2014. doi:10.1001/jamapediatrics.2013.4639.

Author Contributions: Dr O. Andersson had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: All authors.

Acquisition, analysis, or interpretation of data: O. Andersson, Domellöf, Hellström-Westas.

Drafting of the manuscript: O. Andersson, Domellöf, Hellström-Westas.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: O. Andersson, Domellöf, Hellström-Westas.

Obtained funding: O. Andersson, Hellström-Westas.

Administrative, technical, or material support: O. Andersson, D. Andersson, Hellström-Westas.

Study supervision: O. Andersson, Domellöf, Hellström-Westas.

Conflict of Interest Disclosures: None reported.

Funding/Support: This work was supported by grants from the Regional Scientific Council of Halland, common funds for development and research from the Southern Healthcare Region, H.R.H. Crown Princess Lovisa’s Society for Child Care, Uppsala University, and The Framework of Positive Scientific Culture, Hospital of Halland.

Role of the Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: Josefin Roswall, MD, and research nurses Eivor Kjellberg and Monika Nygren, Department of Pediatrics, Hospital of Halland, helped collect the data and received compensation from the grants from the Regional Scientific Council of Halland. The Biostatistical Board, Hospital of Halland, provided valuable statistical advice. We are grateful to the staff at the antenatal units in Halland and at the Department of Obstetrics and Gynecology, Hospital of Halland, Halmstad, and to all the parents and infants who participated in the study.