Delayed versus immediate cord clamping in dichorionic twins <32 weeks: a retrospective study

Abstract Objectives Strong evidence imply that delayed cord clamping (DCC) provides significant benefits for singleton neonates. However, there is little information about the safety or efficacy of DCC in twins to recommend for or against DCC in twins in guidelines. We aimed to determine the effect of DCC on dichorionic twins born at <32 weeks of gestation. Study design This is a retrospective cohort study comparing the neonatal and maternal outcomes of immediate cord clamping (ICC) [<15 second (s)] versus DCC (at 60 s). Generalized estimating equations models were performed accounting for twin correlation. Results A total of 82 pairs of twins (DCC: 41; ICC: 41) were included in analysis. The primary outcome of death before discharge occurred in 3.66% of twins in the DCC group and 7.32% in the ICC group, without a significant difference between the groups. Compared to ICC group, DCC was associated with increased hemoglobin levels [β1 coefficient 6.51; 95% confidence interval (CI) 0.69–12.32. β2 coefficient 5.80; 95% CI 0.07–11.54] at 12–24 h of life. There were no significant differences between the groups in neonatal death, neonatal major morbidities and maternal bleeding complications, although DCC was associated with higher estimated maternal blood loss in the cesarean section group (p = .005). Conclusions DCC for 60 s in dichorionic twins born at <32 weeks of gestation was associated with increased neonatal hemoglobin levels, when compared with ICC. The finding of a higher estimated maternal blood loss by cesarean section in the DCC group calls for further trials to assess maternal safety of this procedure in this patient population.


Introduction
Delaying umbilical cord clamping (DCC) at birth supports a smoother circulatory transition from fetal to neonatal life [1] and increases infants' blood volume [2]. Strong evidence from multiple randomized controlled trials and meta-analyses imply that DCC provides significant benefits for neonates, especially preterm infants [3][4][5]. Thus DCC for preterm infants is consistently recommended by the recent American College of Obstericians and Gynecologists (ACOG) [6] and European Resuscitation Council Guidelines.
The rate of twining has increased largely in recent decades [7]. As twins are at higher risk for prematurity and adverse perinatal outcome than singleton [8], twins may particularly benefit from DCC. However, obstetricians and neonatologists may be hesitant about doing DCC in multiple pregnancies owing to concerns about fetus well-being, unfavorable hemodynamic changes in monochorionic placentation, and risk of maternal hemorrhage. Most studies of DCC usually either excluded multiples or included both multiples and singletons with no stratification analysis of outcomes [9]. One meta-analysis found that none of the four published studies focusing on DCC in multiple gestations stratified outcomes by chorionicity [5]. The conclusions regarding to the neonatal outcomes and maternal safety in preterm twins were varied [10][11][12][13][14]. Statements or guidelines worldwide clearly endorsed DCC for uncompromised preterm infants but most of these guidelines are with no reference to multiple pregnancies [15]. Italian recommendations for placental transfusion weakly recommended DCC for twin newborns from dichorionic pregnancy [16]. No recommendations for or against DCC in multiple gestations were made in the most recent ACOG guidelines [6], Society of Obstetrician and Gynecologists of Canada (SOGC) guidelines [17] and the latest International Liaison Committee on Resuscitation (ILCOR) statement [18] due to insufficient evidence. A further investigation on the safety and efficacy of DCC in multiple gestations is urgently required.
This retrospective cohort study aimed to evaluate the clinical outcomes of DCC in dichorionic twins, born at < 32 weeks of gestation. We hypothesized that DCC for 60 s would not be associated with any adverse neonatal effects or increased maternal hemorrhage risk.

Study oversight
This retrospective cohort study was conducted in Women and Children's Hospital of Chongqing Medical University, which is a tertiary class A hospital with a 126-bed Level 3 neonatal intensive care unit [19]. We routinely admit very preterm infants (VPI) born between 24 /7 and 31 6/7 weeks gestation, which reaches about 220 admissions per year. Our hospital is a national pilot unit for twin pregnancy specialized clinic. We reported the study following the guidelines of Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). This study was approved by the Ethics Committee of the Women and Children's Hospital of Chongqing Medical University (no. 2021015).

Procedures
The umbilical cord was cut within 15 second (s) in the immediate cord clamping (ICC) group and at 60 s after birth in the delayed cord clamping (DCC) group. In 6/2018, a DCC protocol targeted on singleton preterm infants was introduced in this perinatal center. In 1/2019, the DCC protocol was extended to include preterm dichorionic-diamniotic twins based on the Italian guideline with some modifications [16]. The temperature of the delivery room or operating room was pre-adjusted to 28-30 C. Preheated sterile towels were used to dry the newborn infant as quickly as possible and were replaced to prevent heat loss during the intervention. Time was elapsed from birth and called every 15 s by the timer person, who was not part of the resuscitation team. Once the first twin was delivered, he was placed on his mother's perineum in a vaginal delivery (VD) or on his mother's abdomen in a cesarean section (CS). In the DCC group, the attending neonatologist at mother's bedside, initiated the initial resuscitation steps during the first 30 s. At 30 s of life, the neonatologist evaluated heart rate, tone and breathing activity. DCC was discontinued if any of the twins was bradycardic, apneic or gasping at 30 s of life. If the heart rate was >100 bpm and active breathing or efforts to breath were present, the cord was clamped at 60 s. After the cord was cut, the first twin received further resuscitation if necessary following the 2015 American Heart Association Neonatal Resuscitation Guidelines. Giraffe incubator (Giraffe Incubator Carestation SC1,Ohmeda Medical, United states) and Giraffe shuttle(Giraffe shuttle, Ohmeda Medical, United States) were used for resuscitation/transition and intra-hospital transfers.

Inclusion and exclusion criteria
We included all liveborn dichorionic-diamniotic twins born at <32 weeks of gestation and excluded infants with (1) severe placental abruption, (2) major congenital anomalies, (3) general anesthesia, and (4) severe maternal illness that prompted immediate delivery. Twins were excluded from analyses if one of them in the DCC group (1) received less than 60s DCC or (2) had no recorded DCC details.
Maternal outcomes data included estimated blood loss (EBL), postpartum hemorrhage (PPH) defined as EBL >500 ml for vaginal delivery or EBL >1000 ml for cesarean delivery [13], post-delivery decrease in maternal hemoglobin, postpartum infection, blood transfusion and hysterectomy.

Statistical analyses
Maternal and neonatal characteristics and outcomes were compared between the groups. For continuous variables, the Student t test or Mann-Whitney U test where applicable was used. For categorical variables, v 2 or Fisher exact test where applicable was used. Generalized estimating equations models were performed to account for the correlation between twins. Biologically plausible confounding variables including gestational age and birth weight were selected for the statistical model 1. Gestational age, birth weight, mode of delivery, administration of antenatal corticosteriods and maternal diabetes (pre-gestational or gestational diabetes) were selected for the statistical model 2. b coefficients for continuous variables and crude and aOR for categorical variables, along with 95% CIs, were calculated. To determine whether the neonatal outcomes varied by gestational age, planned subgroup analysis was performed among infants born at (29 þ6 weeks vs. those born between 30 þ0 and 31 þ6 weeks. p < .05 or a 95% CI that does not include 1 for ORs and 0 for b coefficients denoted significance, and all tests were 2-tailed. Statistical analyses were performed with SPSS (version 22, IBM, Chicago, IL) and SAS Enterprise Guide software (version 9.21, SAS Institute Inc., Cary, NC) (SAS for GEE model).

Maternal and neonatal demographics
From 2017 to 2020, 105 dichorionic twins pairs were born at <32weeks of gestation and 82 twins pairs were included in our analysis, of whom 41 received DCC for 60 s and 41 received ICC <15 s ( Figure 1). Tables 1 and 2 show maternal and neonatal characteristics. There were significant differences in maternal characteristics including steroids given before delivery (p ¼ .026) and maternal diabetes (p ¼ .003), both of which were included in the adjusted analysis model 2. There were no significant differences in neonatal characteristics except in mode of delivery (p ¼ .001), which was also included in the adjusted analysis model 2.

Maternal outcomes
There were no statistically significant differences in postpartum hemorrhage, postpartum infection and incidence of blood transfusion (Table 3). In the CS group, there was higher estimated maternal blood loss with delayed (p ¼ .005), compared with immediate, cord clamping. 2 women in the DCC group and 6 women in the ICC group, all of whom suffered prenatal anemia, 1 woman with unanticipated placenta accreta in the ICC group, required blood transfusion for symptomatic anemia on postoperative day 1. One woman in the ICC group underwent unplanned hysterectomy.

Neonatal outcomes
As shown in Table 4, the primary outcome of death before discharge occurred in 3.66% of twins in the DCC group and 7.32% in the ICC group, without a significant difference between the groups (aOR1 0.48; 95% CI, 0.08-2.78. aOR2 0.44; 95% CI, 0.05-4.03). The incidence of major neonatal morbidities (NEC, any IVH, RDS, PDA requiring medical or surgical treatment, BPD, EOS and LOS), blood transfusions and duration of mechanical ventilation were similar between the groups. DCC was associated with increased hemoglobin levels (b1 coefficient 6.51; 95% CI 0.69-12.32. b2 coefficient 5.80; 95%CI 0.07-11.54) at 12-24 h in the adjusted analyses compared with ICC. The incidence of PVL was low, which can't be calculated using the adjusted GEE model.
In our a priori planned analyses to determine whether the neonatal outcomes varied by gestational age (Table S1), there were no significant differences in the neonatal death and major morbidities in infants either <30 weeks or ! 30 weeks between the groups (Table S2 and Table S3).

Discussion
There is little information about the safety or efficacy of DCC in twins to recommend for or against DCC in multiple gestations [6]. The current retrospective study of DCC in twins of dichorionic-diamniotic born at <32 weeks of gestation showed no significant difference in the primary neonatal outcome of death before discharge, major neonatal morbidity and maternal hemorrhage, although DCC was associated with increased hemoglobin levels of the infants and higher estimated maternal blood loss in the CS group.
A previous systematic review of preterm singletons showed lower mortality [4,5] and reduced incidence of intraventricular hemorrhage and necrotizing enterocolitis [3] with DCC than ICC. However, the neonatal outcomes of mortality and major morbidity in our study was consistent with existing studies on DCC in twins. An RCT of preterm (28-36 weeks) comparing DCC (24 pregnancies, 51 infants, 30-60 s) to ICC (23 pregnancies, 50 infants, <3-5 s) found no significant differences between the groups in neonatal outcomes, however DCC was associated with a higher PPH rate compared with ICC [12]. A retrospective study of 31 pairs of twins (23 þ0 -31 þ6 weeks) comparing DCC (60 s) to ICC (not defined) found a reduction in RBC transfusions in first week in the DCC group and no differences in neonatal outcomes [10]. Another large retrospective cohort study of 1597 pairs of twins (<30 weeks) comparing DCC !30 s versus <30 s found a reduction of blood transfusions and no difference in neonatal death and/or severe neurological injury [14].  These three prior studies involved monochorionic and dichorionic twins without stratifying by chorionicity. A retrospective study involving 58 pairs of dichorionic twins (23-32 weeks) comparing DCC (30 s; N ¼ 8) to ICC (not defined; N ¼ 50) also did not find significant differences in neonatal outcomes between the two groups. In addition, the Australian Placental Transfusion Study (APTS) including 1566 infants born before 30 weeks of gestation, of which 24.9% were of multiple births, found no significant differences in the incidence of death or major morbidity [9]. Whether DCC in twins reduces mortality or major morbidity requires verification in large randomized trial. The higher hemoglobin level at 12-24 h after adjusting for confounding factors in the DCC group suggest an effective placental transfusion. However, there was no difference in the total number of blood transfusions before discharge. Two previous studies found no differences in hematocrit at birth or in admission [10,12], while the other two previous studies defined no details about the hemoglobin or hematocrit levels [11,14]. As in our study, there seems to be higher hematocrit levels at 12-24 h of life in the DCC group (52.58 ± 5.55) than in the ICC group (50.97 ± 5.86) although with no significant difference. Previous study involving 1566 preterm infants found only a 2.7% difference in peak hematocrit in the first week between DCC and ICC group [9]. Our study was not powered to detect a similar finding due to the small size. Besides, the infant-placental blood volume distribution of normal term infants was 67%-33% at less than 5 s after birth, 73%-27% at 15 s and 80%-20% at 1 min [2]. Infants included in our study were all less than 32 weeks of gestation and the blood volume of these infants may be fewer than that of the term. The cord was clamped at less than 15 s in the ICC group and at 60 s in the DCC group in our study. It is possible that cord clamped at less than 5 s in the ICC group could have resulted more profound significant difference between the two groups. Since onset of infants' ventilation, which was not collected in our study, affect placental transfusion and the inadequacy of ventilation during the delaying procedure in the DCC group could partially explain our negative results.
In our study, there was higher estimated maternal blood loss in the CS group with delayed, compared with immediate, cord clamping, which was contrasted to the previous study [13]. One explanation of the discrepancy may be that the patients received umbilical cord clamping before 30 s in early cord clamping group and at least after 30 s in DCC group in the previous study [13], however we defined DCC as cord clamping at 60 s and ICC as less than 15 s. DCC was not associated with maternal bleeding complications in our study, consistent with previous studies [10,13]. A higher rate of maternal PPH was found in the DCC group, however 3/7 PPH cases were triplets [12]. Therefore, further larger sample size of trials targeted to assess the effect of DCC on maternal safety are needed.
Our study has limitations as a single-center retrospective study and a subsequent randomized controlled trial is planned. There were significant differences in mode of delivery, administration of  antenatal corticosteriods and maternal diabetes, which has impacts on the prognosis of the infants. Although we stratified by these potential confounding factors, there may still be some bias in our study. Also there may be selection bias related to implementing DCC. Our study was limited by its small sample size and low numbers of extremely low birth weight infants. Our study was not powered to demonstrate differences in neonatal benefits previous detected in singletons. As there is potential risks of acute twin to twin transfusion syndrome during DCC in monochorionic multiple gestations [6,24], our DCC protocol only includes dichorionic twins but excludes monochorionic twins, which partly contribute to the small sample size of our study. Despite these limitations, we believe it is important to share our findings, as an important gap in the practice of DCC in preterm twins need to be addressed.
In conclusion, DCC for 60 s in very preterm dichorionic twins was associated with increased neonatal hemoglobin levels, when compared with ICC. The finding of a higher estimated maternal blood loss by cesarean section in the DCC group was unexpected. Further larger clinical trials are urgent needed to confirm the observed findings and long term neurodevelopmental outcomes.

Disclosure statement
No potential conflict of interest was reported by the author(s).   Generalized estimating equations models were performed to account for the correlation between twins. Gestational age and birth weight were selected for the statistical model (1) Gestational age, birth weight, mode of delivery, administration of antenatal corticosteriods and maternal diabetes were selected for the statistical model (2), can't be calculated for the low incidence. Ã Statistically significant.