Received Date : 06-Apr-2016

Revised Date : 22-Jun-2016

Accepted Date : 24-Aug-2016
Article type : Regular Article
Accepted Article
Randomised control trial showed that delayed cord clamping and milking resulted in

no significant differences in iron stores and physical growth parameters at one year

of age

Short title: Placental redistribution comparison

Authors: Agarwal S1; Jaiswal V1; Singh D1; Jaiswal P2; Garg A3; Upadhyay A1

Department of Pediatrics1; LLRM Medical College, Meerut; UP, India; Department of

Pediatrics2; PGIMER, Dr. Ram Manohar Lohia Hospital, New Delhi, India; Department
of Microbiology3; LLRM Medical College, Meerut; UP, India.

Corresponding Author:

Dr. Shivam Agarwal

Department of pediatrics
L.L.R.M. Medical college, Meerut, (India)
Tel: +91-9456230221
email: drshivam86@gmail.com

Abstract

Aim Placental redistribution has been shown to improve haematological outcomes in the

immediate neonatal period and early infancy. This study compared the effects of delayed

cord clamping (DCC) and umbilical cord milking (UCM) on haematological and growth

parameters at 12 months of age.

Methods This was a follow-up study of a randomised control trial, conducted in a tertiary

care paediatric centre from August 2013 to August 2014. We studied 200 apparently healthy

Indian infants randomised at birth to receive DCC for 60-90 seconds or UCM. The outcome
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/apa.13559
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 measures were iron status and physical growth parameters at 12 months.

Results Of the 200 babies, 161 completed the follow up and baseline characteristics were
Accepted Article
comparable in both groups. The mean haemoglobin in the DCC group (102.2 (17.2) g/L and

serum ferritin 16.44 (2.77) µg/L) showed no significant differences to the UCM group (98.6

(17.1) g/L and 18.2 (2.8) µg/L) at one year. In addition, there were no significant differences

in weight, height and mid upper arm circumference in the two groups.

ConclusionTerm-born Indian infants who had DCC at 60-90 seconds or UCM showed no

significant differences in ferritin and haemoglobin levels and growth parameters at 12

months of age.

Keynotes:
 lacental redistribution has been shown to improve haematological outcomes

in the immediate neonatal period and early infancy. 

 We followed up 161 term-born Indian infants who had taken part in an earlier

randomised control trial to compare delayed cord clamping at 60-90 seconds

and umbilical cord milking.


 The two placental redistribution techniques resulted in no significant

differences in iron stores and physical growth parameters at 12 months of

age. 

INTRODUCTION

Anaemia due to iron deficiency is a major health problem in infancy especially in low-income

countries (1). According to the third Indian National Family Health Survey, 70% of infants

between six and 11 months of age were found to be anaemic (2). The development of brain,

including myelination, dendritogenesis, functioning of the neurotransmitter, glial and

neuronal energy metabolism, depend on iron and its deficiency has been associated with

impaired motor and behavioural development (3–5). In an attempt to prevent iron deficiency

anaemia during infancy, enteral and parenteral iron supplements have been tried. Although

enteral iron is the predominant mode of iron supplements, it is ineffective due to cost,

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 unavailability and poor compliance in resource poor settings. Free radical injuries to mucosa

are also an issue. Parenteral supplements are unsafe, as there is a risk of iron overload and
Accepted Article
anaphylaxis. Therefore it is prudent to have a safe, economical, feasible, more practicable

and less time consuming intervention to prevent iron deficiency anaemia.

Immediately after birth, the newborn infant still shares its blood circulation with the placental

unit through the umbilical vessels. If immediate cord clamping takes place, the blood in the

fetal/placental unit is distributed in a ratio of approximately 2:1, while a delay in clamping for

three minutes provides an additional blood volume of 15-35ml kg-1 of body weight (6,7). This

placental transfusion has been shown to result in increased haemoglobin and haematocrit in

the immediate neonatal period (8,9). Two interventions related to placental redistribution are

delayed cord clamping (DCC) and umbilical cord milking (UCM). These two techniques have

been shown to improve the haematological status in preterm and term infants at four to six

months of life (10–13). Based on the available studies, the American Academy of Pediatrics

has recommended delayed cord clamping in most vigorous term and preterm newborns, as

it reduces anaemia, improves iron status and reduces the need for a blood transfusion (14).

There have been trials to assess the effects of DCC on its own and of delayed versus early

cord clamping on haematological parameters in late infancy, but none of them have

compared the effects of DCC versus UCM on infant health and iron status in late infancy

(1,11). Therefore, we designed a follow-up study to compare the effects of DCC at 60-90

seconds and UCM on haematological and growth parameters in infants at 12 months of age.

METHODS

This follow-up study of the original randomised controlled trial, which has previously been

described (15), was conducted at LLRM Medical College, Meerut, a tertiary care hospital in

North India, between August 2013 to August 2014. The study was approved by the

Institutional Ethical Committee for the College. Full-term newborns with a gestational age of

37 to 41 weeks were randomised into the UCM or DCC groups if they fulfilled the inclusion

criteria (15), namely that the mother was healthy, was a non-smoker and had an

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 uncomplicated pregnancy with an expected vaginal delivery (Figure S1). The 1:1

assignments were contained in sealed, numbered, opaque sealed envelopes that

Accepted Article
randomised the infant to delayed cord clamping 60-90 seconds after delivery or umbilical

cord milking less than 30 seconds after delivery. The envelope was opened by the staff on

duty after ensuring the baby was vigorous and had no knots in the cord, with an appropriate

cord length of 25cm. The clinicians could not be blinded to the randomisation as they

performed the interventions, but all the staff and researchers involved in noting the growth

parameters and performing the haematological tests were blinded to the allocation group.

In the UCM group, the umbilical cord was cut and clamped at the placental end, 25cm from

the umbilical stump, as soon as possible to avoid placental transfusion before clamping.

Cases were excluded from the study if the time exceeded 30 seconds. The umbilical cord

length was measured using the 25cm sponge holding forceps used for clamping. The

umbilical cord was raised and milked from the cut end toward the infant three times at 10cm

s-1 and then clamped 2-3cm from the umbilical stump. In the DCC group, the obstetrician

was informed about the intervention to delay the clamping for 60-90 seconds, which was

done by using the wall-mounted clock in the delivery room.

The newborn infants randomised at birth to the two intervention groups were followed up at

one year of age. The primary outcomes of our study were haematological parameters,

namely haemoglobin, haematocrit and serum ferritin levels, at 12 months of age. Telephone

reminders were used to ensure that the parents attended the follow-up visits. The wellbeing

of the baby was discussed and the parents were also reminded about the visit to the hospital

for immunisation and follow up. Before the visit, the parents were asked to complete a

questionnaire to assess the adequacy of feeding. Anthropometric measurements for weight,

height and mid upper arm circumference were taken at the follow up and blood samples

were drawn to assess the haematological parameters at 12 months of age. The means and

standard deviations (SD) of the age group were calculated for both of the intervention

groups, which were 11.1 (1.3) months and 11.1 (1.2) months in the DCC and UCM groups

respectively. Venous blood sampling was performed after the application of an EMLA local

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 anaesthetic (AstraZeneca, Cambridgeshire, United Kingdom). Blood was collected in

ethylene diamine tetra acetic acid tubes for haemoglobin, haematocrit and in plain vials for
Accepted Article
serum ferritin. Haemoglobin was measured by the cyan method, haematocrit by the

Wintrobe method and serum ferritin was estimated using the micro enzyme-linked

immunosorbent assay technique with a ferritin kit (bioMérieux, Marcy l'Étoile, France). The

feeding history was taken from the infant’s parents at the time of follow up, using a

questionnaire that covered breast feeding duration, feeding other milk to the baby than

breast milk before six months of age, type of complementary feeding, types of feeds and

amount of feeds. A separate questionnaire covered prior hospitalisation for more than 12

hours and intake of iron supplements, namely iron tablets, preparations or formula

containing iron for at least 30 days, was considered. The morbidity profile of the infants was

also recorded in terms of fever episodes, defined as an axillary temperature of more than

99°F. The standard World Health Organization (WHO) definitions were used for diarrhoea,

pneumonia and exclusive breast feeding (16-18). A subgroup analysis was carried out for

small for gestational age babies, defined as a weight of less than the 10th centile for age and

sex nomograms (19).

The following definitions were used in the study: anaemia was a haemoglobin of < 10.7 gm/

dl at 12 months of age and for iron deficiency, a serum ferritin of < 10.9 µg/dl was

considered significant (20). The secondary outcomes were growth parameters in the two

groups. Anthropometric measurements were carried out at the 12-month follow up. Weight

was measured by using a Dolphin DT-30k electronic weighing machine (Dolphin, New Delhi,

India) with a precision error of 5g. The length was measured using an Harpenden’s

infantometer (Chasmors Ltd, London, United Kingdom) designed to measure lengths

between 0-100cm with a precision of 1mm. Head circumference was measured using a self-

retracting, 0.7cm wide, flat metal tape with black lead-in strip, with a range of 0-200cm,

calibrated to 1mm. All data was recorded by a single investigator (SA). Z scores for weight

for age, weight for length, length for age and head circumference for age were calculated

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 using version 3.2.2 of the WHO anthropometric calculator, downloaded from

www.who.int/childgrowth/software/en/.
Accepted Article
Statistical analysis

We compared groups with continuous variables with normal distributions using the Student’s

t- test and the Mann-Whitney U test was used for variables with skewed distribution. We

estimated the 95% confidence intervals (95% CI) using the Hodges and Lehmann estimator.

Serum ferritin was not normally distributed and was skewed to the left. However, on log

transformation, it was converted to a normal distribution and the antilog was taken.

Categorical variables were compared between the groups using the chi-square test or

Fisher’s exact test as applicable. The analysis was carried out using STATA 12 software

(StataCorp LP, Texas, USA). Confounding factors were clinically chosen and no sensitivity

analysis was performed before choosing them. The effect of confounding factors was

analysed using multivariate logistic regression.

RESULTS

Of the 200 newborn infants enrolled in the trial, 161 finally underwent haematological and

anthropometric examinations at 12 months of age (Figure S1), with 78 (48.4%) in the DCC

group and 83 (51.6%) in UCM group. The mean age at follow up was comparable in the two

groups (p>0.05). Other baseline characteristics were also comparable in the two groups

except for maternal haemoglobin (Table S1). The baseline clinical characteristics of the

babies who were lost to follow up were comparable to those who completed the full follow

up.

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 The adjusted mean (SD) for haemeoglobin, haematocrit and the median and interquartile

range for ferritin showed no significant differences in the DCC and UCM groups. As
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previously reported by Jaiswal et al, there was no significant difference between the

haemoglobin, haematocrit and serum ferritin in the two groups at 48 hours and at six weeks

of follow up (Table S2). The mean haemoglobin at 48 hours was higher in the small for

gestational age babies than the appropriate size for age babies (p = 0.04). However, the

mean haemoglobin and serum ferritin levels were comparable in the two groups at six weeks

of age (p = 0.12) (15). There was no significant differences in growth parameters (Table S3),

mean haemoglobin, haematocrit and serum ferritin in the two groups at 12 months of age (p

= 0.92)

DISCUSSION

This study demonstrated that the difference between haemoglobin and serum ferritin levels

was insignificant after UCM or DCC at 60-90 seconds at 12 month of age. Also, the

difference in the two groups was insignificant for weight, length and head circumference.

Incidence of wasting and stunting were comparable in the two groups.

The effects of DCC and UCM have been well documented in previous studies. Data

accumulated to date shows that DCC, when compared to early cord clamping, improved

haematological outcomes with respect to haemoglobin and serum ferritin at two to six

months of age, but not later in term (1,9,10,12,13,21–23) and preterm neonates (24). DCC

has also been reported to improve the neurodevelopmental outcome at four years of age

(25), but have comparable effects to UCM in preterm neonates (26). UCM also resulted in

improved haematological status in term (8) and preterm infants (24). Only two studies have

compared the effects of UCM with DCC, but both were only in preterm neonates (24,27).

Katheria et al demonstrated higher systemic blood flow with UCM, based on four strippings,

in preterm neonates compared with DCC at 45-60 seconds in Caesarean deliveries, but not

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 in vaginal deliveries, Neonates undergoing UCM also had higher haemoglobin levels,

delivery room temperature and blood pressure over the first 15 hours and urine output in the
Accepted Article
first 24 hours of life (27), Rabe et al concluded that milking the cord four times achieved a

similar amount of placento-fetal blood transfusion compared with delaying clamping at 30

seconds (24).

It has been estimated that the total blood volume in the fetal/placental unit is approximately

120 ml kg-1 of fetal weight. The distribution of the blood in the fetus and placenta after

immediate cord clamping has a ratio of around 2:1 (10). Allowing placental transfusion to

occur for at least three minutes, leads to 15-35 ml kg-1 of blood transfusing into the baby,

with only 15 ml kg-1 remaining in placenta after complete transfusion (6,28). Theoretically

this should lead to sufficient iron stores in the infant to meet its iron requirements for more

than three months (29).

There have been no studies in the literature that have compared the effects of DCC and

UCM on term neonates and followed them until late infancy. Theoretically, DCC for a longer

period of time should lead to the passage of more blood transcord than milking with early

cord clamping. This is because during DCC the blood is being transferred from the placenta

towards the baby, facilitated by uterine contractions and gravity, while in UCM with a cut

cord, the placental unit is separate and only the blood stored in the cord can be passively

transferred. However, this was not observed in our study and the two groups showed no

significant differences in ferritin and haemoglobin levels, as well as in growth parameters, at

12 months of age. However the p value between the groups when comparing haemoglobin

and hematocrit were between 0.05 and 0.10, which were close to significant (Table S2).

According to the third Indian National Family Health Survey, nutritional deficiencies in India

are evident from the time of birth and the incidence of stunting and underweight rise rapidly

in the first two years of life. The proportion of children who are stunted rises sharply from

birth to 38% at 12 months of age and peaks at 59% at 20 months of age (2). However, in our

study, 5.5% of infants who received DCC at 60-90 seconds and 9.6% infants who received

UCM were stunted. At 12 months of age, 14% and 7% of the children in the DCC and UCM

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 groups were wasted, respectively. The proportion who were underweight had a similar, but

lower, pattern of fluctuation than observed for the subjects who were stunted.
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According to the National Family Health Survey, the prevalence of anaemia was 70% in

children aged 6-59 months (2). Children aged 6-30 months had the lowest haemoglobin

concentrations (30). In our study, about 60% of infants were anaemic, while approximately

30% had iron deficiency. The WHO definition for anaemia is a haemoglobin of < 110 g/L and

for iron deficiency anaemia it is a ferritin level of < 12 µg/L in children under the age of five.

So the cut-off values for haemoglobin and serum ferritin to diagnose iron deficiency and iron

deficiency anaemia in infants at 12 months of age are not well defined. We use the cut-off

values provided by Neve Vendt et al, which were a haemoglobin of < 107 g/L, with a

sensitivity of 67% and specificity of 87%, for iron deficiency anaemia and ferritin levels of <

10.7 µg/L, with a sensitivity of 83% and specificity of 80% (20).

The strength of this study was that it followed up a randomised control trial with a reasonable

sample size and a good follow-up rate. This was probably the first study to directly compare

the effect of DCC at 60-90 seconds and UCM in term neonates in late infancy. We measured

serum ferritin as it reflected the accurate level of the infant’s iron stores along with

haemoglobin in late infancy. One limitation of our study was related to the milking technique.

We cut the cord and then milked it and that may have limited the refilling of the cord from the

placenta. In addition, in the DCC group, cord clamping could have been delayed a bit more,

for two to three minutes instead of 60-90 seconds, to increase the amount of placental

transfusion. Moreover, the study of neurodevelopmental outcome could have been

improved, as measuring the outcome at four years was shown to improve the

neurodevelopment in a DCC group (25).

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 CONCLUSION

Before we carried out this study we knew that DCC and UCM improved haematological
Accepted Article
parameters for neonates, which is important in populations with increased risks of neonatal

and childhood anaemia. Our findings show that carrying out UCM or DCC at 60-90 seconds

showed no significant differences in ferritin and haemoglobin levels as well as in growth

parameters in infants at one year of age. As DCC has already been recommended as

standard care in most deliveries by the American Academy of Pediatrics, we suggest that

UCM can be used in term neonates if DCC is not feasible. In cases where the neonate

requires resuscitation, then UCM can be tried for better neonatal outcomes. Further studies

with UCM with the attached placenta should be carried out and followed until late infancy to

assess its effects and persistence.

ACKNOWLEDGEMENTS

Our thanks go to Mr C. P. Yadav from the Department of Biostatistics at the All India

Institute of Medical Sciences for his help with the statistics and the resident doctors in the

Department of Pediatrics for their valuable support.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare.

FINANCE

This study did not receive any external funding.

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 Abbreviations:

DCC – Delayed cord clamping

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WHO – World Health Organization

SD – Standard deviation

UCM – Umbilical cord milking

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