Effect of Time of Cord Clamping on Serum Bilirubin Level among Full Term Babies Born at Tikur Anbessa Specialized Hospital: A Three Arm Randomized Control Trial.

Background: Delayed cord clamping (DCC) above one minute is associated with nutritional and developmental benets newborns. DCC could also lead to elevated serum bilirubin, leading to jaundice that require phototherapy at the early ages of life. However, evidence on the relationship between DCC and outcomes is limited in resource-limited settings. Objective: To evaluate the effect of the time of cord clamping on serum bilirubin level at 24 hours post-birth in an Ethiopian Tertiary Care hospital. Methods: A three-arm, single-blind, randomized controlled trial was conducted between October 2019-January 2020 targeting healthy, full-term neonates. Study participants were randomly assigned into one of the following interventions depending on the timing of cord clamping: immediate cord clamping (<30 seconds), intermediate cord clamping (60 sec), and delayed cord clamping (180 seconds). The primary outcome is serum bilirubin level in the newborns, which was assessed at 24 - hour after birth and before they were discharged home. Descriptive analysis was applied to assess differences between groups in terms of demographic, obstetric, and social characteristics of participants, while linear regression model was employed to examine the association between cord clamping time and serum bilirubin level. STATA version 14 was used for statistical analyses. Result: A total of 141 term newborns were included and randomized into the three categories. Demographic, social and obstetrics factors of the women were not different across groups except labor duration that had a P-value < 0.05. Time of cord clamping was not a signicant predictor of total serum bilirubin levels at 24 hours post-birth, whereas cord blood total serum bilirubin (coef. 0.24; p<0.05) and bilirubin nomogram high-risk zone (Coef. 6.25; p<0.001) were signicant predictors. Conclusion: Time of umbilical cord clamping has no effect on the total serum bilirubin level of neonates at least within 24 hours of birth.


Introduction
According to global estimates, Ethiopia is one of the top nine countries that contribute half of the total childbirth around the world with more than 9000 births every day. 1 From the total live births, around 75% of them are delivered with spontaneous vaginal delivery under the risk of facing several challenges and complications around time of birth. 2 Jaundice (hyperbilirubinemia) is one of the challenges within the rst 24 hours of childbirth.
Hyperbilirubinemia is de ned as an excessive level of bilirubin (>34.2 μmoL/L or 2 mg/dL) either in the conjugated or unconjugated form. 3 Eighty percent of bilirubin is derived from the heme group of hemoglobin, which comes from the destruction of red blood cells (RBCs) in the reticuloendothelial of the liver, spleen, and bone marrow. 4 The destruction of RBCs could occur either due to premature rupture or excessive RBCs (polycythemia) and is directly in uenced by the delay in the clamping time that gives extra 80-100mL of blood within the rst 1-3 minutes to enter the neonate circulation. [5][6][7] The case fatality rate for neonatal jaundice and severe complications in Low-and Middle-Income Countries range between 20% and 60%. 8 The case in Ethiopia is not different from the rest of Africa; it is estimated that about 13% of neonates born in hospitals develop neonatal jaundice, while 7% of them develop severe related complications like bilirubin encephalopathy. 9 During the rst few minutes after birth, there is a circulation of blood from the placenta to the infant.
Delaying clamping of the umbilical cord by 2 to 3 minutes or until the pulsation of the cord stops will allow the physiological transfer of placental blood to the infant. 3 Evidence shows that delayed cord clamping is bene cial in preventing neonatal hospital mortality. 10 This is mainly because of its potential to lower the risk of reduced hematocrit level at 24 hours and also reducing the risk of iron de ciency at 3-6 months post-birth. 10,11 The implementation of delayed cord clamping (DCC) has a good deal of support globally for the healthy outcome of neonates and mothers, despite arguments on its potential adverse effects.
Although multiple studies revealed the immediate and long-term bene ts of implementing DCC for both the neonate and the mother, 3 emerging anecdotal evidence reported the counter effect of delaying the cord clamping; mainly attributed to the increasing neonatal risk of polycythemia and neonatal jaundice that require phototherapy. 11,12 Based on the ndings from various randomized clinical trials and reviews, the World Health Organization (WHO) recommends delayed umbilical cord clamping (not earlier than 1 min after birth), as it can improve maternal and infant health and nutrition outcomes. 3 However, there is a need for studies to establish the association between DCC and risk of hyperbilirubinemia on newborns. 3 Particularly, there is a relative paucity of data from resource-limited settings on the clinical outcomes associated with the implementation of delayed clamping. Therefore, creating an evidence based on the effect of DCC on outcomes in newborns will be instrumental in informing clinical guidelines.
This study aims to evaluate the effect of time of umbilical cord clamping on serum bilirubin level and proportion of hyperbilirubinemia that requires phototherapy.

Study setting
This three-arm parallel randomized control trial was conducted at Tikur Anbessa Specialized hospital (TASH)-the largest referral and teaching hospital in Ethiopia. The study protocol (attached as appendix) was approved by the institutional review board at the College of Health Sciences, Addis Ababa University (AAU/SPH-IRB/0056) and respective departments of obstetrics/gynecology and neonatology with in the TASH. written informed consent was provided by the participants before they were randomized in the study.

Study population and recruitment
The study participants were pregnant women with fetuses that are scheduled for a spontaneous vaginal delivery and with an inclusion criterion of; a gestational age of ≥ 37 weeks as diagnosed with rst day of last menstrual period (LMP) or ultrasound and those with no gross complications related with the pregnancy and delivery, including mal-presentation, fetal distress, and congenital malformations. Women at greater risk of adverse effects due to delayed clamping were excluded from the study. The main exclusion requirements were those with previa or placenta abruption, early diagnosed fetal anomalies, those with fetal anemia, those with pre-eclampsia or signi cant maternal anemia. 13 Participants were recruited in the period between October 2019 and January 2020.

Randomization and interventions
The eligible participants were initially approached by the principal investigator (who is a research midwife) to obtain consent before or on the day of delivery. The consented participants were randomized into three groups in a 1:1:1 ratio using pre-determined six block randomization sequences.
The interventions the different groups received are as follows: Group 1: clamping of the umbilical cord immediately (<30seconds) after birth.
Group 3: clamping of the umbilical cord time was ≥ 180 seconds (3-minutes) after birth.
The allocation of the participants was performed after obtaining their informed consent earlier in the late rst stage labor (i.e.≥8cm cervical dilatation). The principal investigator (PI) prepared the allocation codes with sealed, opaque, and identically colored envelopes initially at the beginning of the study. One of the co-investigators, who was blinded to the participant screening and randomization process, was responsible for opening the envelope and revealing the nature of intervention to the birth attendant when the mother enters the second stage of labor or was ready for childbirth proper.

Outcomes
The primary outcome is the level of total serum bilirubin of the newborns at the 24 hours of age and the secondary outcome is the proportion of neonates that developed hyperbilirubinemia before discharge to home.

Data collection procedure
The data collection had two phases. First, around time of birth; and both maternal and fetal characteristics were recorded. Second, at the age of 24 hour after delivery. Blood samples for the primary outcome were collected from veins and were transported to the laboratory with anti-coagulated test tube by one of the research investigators within 30 minutes of the sample collection. The source of sociodemographic and obstetric data was the medical record chart of both the mother and the newborn.
The rest of the data was obtained from direct interview of the mother and laboratory ndings. The sample size which was calculated using two independent proportions with a power of 90% and α=0.05 resulted in a total of 141 participants that were subsequently categorized into three arms (47 in each group).
The data collectors discussed with eligible participants and/or their companions about the procedure. The discussion includes explaining the risks and bene ts associated with the interventions later in the rst stage of labor. Informed consent was then secured either from the participant or respective companions before the second stage labor ensued or upon preparing the mother for delivery. When the mother is ready for delivery, the randomly assigned sealed-envelope containing the group assignment. The investigator monitored the clock with a digital second counter to inform the birth attendant when the time for that particular intervention ended.
After expulsion of the newborn, the birth attendant had to collect one milliliter cord blood and handed it to the assistant after separating the cord irrespective of the group assignment. The collected blood samples were then stored in a test tube, transported to the assigned laboratory. The results were recorded on the newborn chart by the attendant according to the hospital protocol.

Statistical analysis and data management
After data collection, the completeness and consistency of the questionnaires were checked. Then both the questionnaires and the variables coded, entered into the Epidata manager version 4.4.2.1 (Epi-Data Association, Denmark) and the necessary data cleaning performed.
Descriptive analysis was performed to compare baseline characteristics of the participants and reported using mean and standard deviation (SD) for discrete continuous data types. Ina addition, frequencies and percentages used for categorical data presentation. Pearson's chi-square test was used to compare categorical variables across the different cord clamping times, and Fisher's exact test used when there were less than ve observations within the groups. Kruskal Wallis rank test was also used for comparison of continuous variables based on the time of clamping. We used unadjusted linear regression model to evaluate the effect of times for cord clamping on serum bilirubin level and multiple linear regression to conduct an adjusted analysis controlling for other important factors. Associations were considered signi cant at p<0.05 and analysis was performed using STATA version 14 software (Stata Corp LLC, College Station, TX, USA). Table 1 presents the basic demographic, social and obstetric characteristics of participant mothers. Most of the participants (102; 72%) aged between 20-34 years, while (29; 20%) of them were under the age of 20 years. A total of 107 (75.88%) participants have had the recommended antenatal visit of more than four times. From total participants, 127 (90.07%) had normal and 14 (9.93%) prolonged labor. The association between maternal and obstetrics factors were also compared based on the different time of umbilical cord clamping ( Table 2). None of the factors showed a signi cant difference across the different cord clamping times except the duration of labor, which is signi cantly longer in the intermediate clamping time (P-value of < 0.05) compared with the immediate or delayed clamping groups.  Table 3 presents the association between neonatal factors and time of cord clamping. The gender distribution is similar across the three groups. While most of the newborns (94.3%) were fed with breastmilk only, this did not vary based on the time of cord clamping. APGAR score was also not signi cantly associated with the umbilical cord clamping time.

Results
Majority of the newborns (95.74%) were in the low-risk zone of the bilirubin nomogram within the rst 24 hours of their birth and only three (2.12%) newborns required phototherapy within 48-72 hours post-birth, albeit this was not signi cantly related with time of cord clamping. The mean weight of neonates during birth was 3251gm (±60), 3259gm (±63), and 3247gm (±67) for immediate (<30 sec), intermediate (30 -60sec) and delayed (≥180sec) umbilical cord clamping times, respectively. Further, there were no variations in average temperature, and cord blood RBC and TSB between groups. Unadjusted linear regression of total serum bilirubin amount with umbilical cord clamping time revealed a negative but non-signi cant association between the two variables (Coef. -0.15; P-value = 0.19). This is presented in Table 4 below. Multiple regression analysis revealed that bilirubin nomogram high-risk zone (p < 0.001) and cord blood bilirubin (p< 0.05) were signi cant predictors of serum bilirubin levels within 24 hours of birth and delaying cord clamping time to three-minute shows no signi cant association with the total serum bilirubin (TSB) levels within 24 hours of birth.

Discussion
This study tested the hypothesis that delayed and immediate umbilical cord clamping would have a similar predictive effect on TSB levels of healthy full-term newborns within 24 hours of birth. Our ndings indicate that the time of umbilical cord clamping did not affect TSB levels that is measured at 24 hours after birth. This nding implies supports that the arguments on the safety of DCC unlike previous ndings reporting delayed time of cord clamping is associated with increased serum bilirubin and related jaundice and its complications. [14][15][16][17] A study conducted on effects of DCC on residual placental blood volume, hemoglobin and bilirubin levels in term infants indicated that there was a signi cant increase in the number of neonates required phototherapy when there was a delay in the umbilical cord clamping time to 3 minutes. 11 On the contrary, our nding indicates that there were only three newborns (one in each arm) that develops jaundice requiring phototherapy. This may be attributed to the fact that in our study serum bilirubin levels measured within 24 hours after birth and that can minimize the chance of capturing the late (48-72 hours) elevations in serum bilirubin levels.
Elevated cord blood RBC level or polycythemia was found to be a signi cant predictor of elevation of TSB around the early hours (24-72 hours) of life. 12,18 Further, cord blood TSB was also signi cantly associated with serum bilirubin levels after 24 hours of birth. On the other hand, the cord blood RBC was not different from the normal range in any of the groups, which implies that an umbilical cord clamping time should not be considered as something detrimental to newborns health.
In our study, Bilirubin Nomogram high-risk zone was strongly associated with TSB levels within 24 hours of birth. This is different from what was reported previously by Judith et al 11 that found an insigni cant relationship between these variables. This discrepancy could be attributed to the fact that our study had only one participant in the high-risk zone. Nevertheless, using Bilirubin Nomogram is believed to be a gold-standard measurement to clearly predict, monitor and manage elevated bilirubin level at the early hours of the newborn before jaundice and further complications developed. 19,20

Conclusion
This research nding found out and concluded that time of umbilical cord clamping will have no signi cant relationship with the total serum bilirubin levels of neonates at least within 24 hours of birth. In addition, delaying an umbilical cord clamping time to three minutes had no effect on elevating the total serum bilirubin levels of neonates that can put them in danger of jaundice requiring phototherapy and it can be considered as an easy and safe procedure that have a multifaceted importance for the newborns.  Figure 1 Sampling technique of study participants from mothers came for child birth at TASH from October 2019 -January 2020.

Figure 2
Relationship between time of cord clamping and total serum bilirubin level at the age of 24 hours of birth among neonates born at TASH from a period of October 2019 -January 2020.