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Neonatal reticulocyte count during the early postnatal period

  • Mika Yamada
    Affiliations
    Department of Pediatrics, Fukushima Medical University School of Medicine, Fukushima, Japan
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  • Mina Chishiki
    Affiliations
    Department of Pediatrics, Fukushima Medical University School of Medicine, Fukushima, Japan
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  • Yuji Kanai
    Affiliations
    Department of Pediatrics, Fukushima Medical University School of Medicine, Fukushima, Japan

    Department of Pediatrics, Ohta General Hospital Foundation Ohta Nishinouchi Hospital, Koriyama City, Fukushima, Japan
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  • Aya Goto
    Affiliations
    Health Information and Epidemiology Center for Integrated Science and Humanities, Fukushima Medical University School of Medicine, Fukushima, Japan
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  • Takashi Imamura
    Correspondence
    Corresponding author. Department of Pediatrics, Ohta General Hospital Foundation Ohta Nishinouchi Hospital, 2-5-20, Nishinouchi, Koriyama City, Fukushima, 963-8558, Japan.
    Affiliations
    Department of Pediatrics, Ohta General Hospital Foundation Ohta Nishinouchi Hospital, Koriyama City, Fukushima, Japan
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Open AccessPublished:May 04, 2020DOI:https://doi.org/10.1016/j.pedneo.2020.04.004

      Background

      There are few reports on reticulocyte count during the early postnatal period, and its clinical significance is not well understood. To examine the relationships between neonatal reticulocyte count and other perinatal variables.

      Methods

      We conducted a retrospective cohort study of neonatal infants who were admitted to the neonatal intensive care unit (NICU) of Ohta Nishinouchi Hospital, Japan, between April 1, 2016 and July 31, 2019. All blood samples were collected within 3 h after admission.

      Results

      Four hundred and twenty-eight infants were included, of whom 317 (74.1%) were preterm and 111 (25.9%) were term. Two hundred and ninety-nine infants (69.9%) were born by cesarean section. The median reticulocyte counts (‰) for all gestational ages (GAs) were as follows: 24–25 wks (n = 11), 124.1 (range: 106.3 to 148.6); 26–27 wks (n = 25), 111.1 (range: 55.5 to 144.3); 28–30 wks (n = 52), 81.9 (range: 35.6 to 131.5); 31–33 wks (n = 86), 71.6 (range: 28.3 to 116.6); 34–36 wks (n = 143); 59.6 (range: 30.2 to 110.9); and 37–41 wks (n = 111), 43.2 (range: 21.9 to 69.2). There were significant relationships between the neonatal reticulocyte count and gender [p < 0.01, odds ratio (OR), 0.37; 95% confidence interval (CI), 0.21 to 0.64], GA (p < 0.01, OR, 0.92; 95% CI, 0.90 to 0.93), delivery type (p = 0.03, OR, 0.51; 95% CI, 0.28 to 0.95), maternal haemoglobin before delivery (p < 0.01, OR, 0.74; 95% CI, 0.60 to 0.91), tracheal intubation at resuscitation (p = 0.04, OR, 2.75; 95% CI, 1.04 to 7.32) and mean platelet volume (p < 0.01, OR, 0.51; 95% CI, 0.35 to 0.74).

      Conclusion

      A higher neonatal reticulocyte count in NICU infants may be one of the physiological responses to a more rapid environmental change during the early postnatal period.

      Key Words

      Abbreviations

      ACT
      antenatal corticosteroid therapy
      BT
      body temperature
      BW
      birth weight
      CI
      confidence interval
      CK
      creatine kinase
      CPAP
      continuous positive airway pressure
      C-section
      cesarean-section
      FiO2
      fraction of inspiratory oxygen
      GA
      gestational age
      GDM
      gestational diabetes mellitus
      Hb
      haemoglobin
      HDP
      hypertensive disorder of pregnancy
      IDA
      iron deficiency anemia
      LDH
      lactate dehydrogenase
      Mg
      magnesium
      MPV
      mean platelet volume
      NICU
      neonatal intensive care unit
      OR
      odds ratio
      pH
      hydrogen ion concentration
      PPV
      positive pressure ventilation
      RBC
      red blood cell
      SGA
      small for gestational age
      UCM
      umbilical cord milking
      UmApH
      umbilical artery hydrogen ion concentration
      WBC
      white blood cell

      1. Introduction

      Reticulocytes are the earliest red blood cells (RBCs) observed in the peripheral blood without nuclei, and they comprise 0.6%–2.9% of the RBCs in adult blood and 1.7%–5.0% of those in umbilical cord blood.
      • Paterakis G.S.
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      Flow-cytometric analysis of reticulocytes in normal cord blood.
      One characteristic of reticulocytes is that they only circulate for 24–48 h in the peripheral blood and, unlike ferritin, are not influenced by infection or inflammation. They are considered to be a useful indicator of iron deficiency anemia (IDA).
      • Braekke K.
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      Oxidative stress markers and antioxidant status after oral iron supplementation to very low birth weight infants.
      As they can be examined using only a small amount of blood, reflect bone marrow hematopoiesis with good sensitivity and indicate iron deficiency at an early stage, they have received a good deal of attention. Reticulocyte haemoglobin (Hb) content is also used as an indicator of IDA in low birth weight (BW) and preterm infants.
      • Lorenz L.
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      • et al.
      Reticulocyte haemoglobin content as a marker of iron deficiency.
      However, there are few reports on the reticulocyte count in the early postnatal period, and its clinical significance is not well understood. On the other hand, nucleated erythrocytes have been reported to be elevated in cases of neonatal asphyxia,
      • Fox H.
      The incidence and significance of nucleated erythrocytes in the foetal vessels of the mature human placenta.
      and hypoxemic stimulation before birth is known to be involved in erythropoiesis.
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      Regulation of erythropoiesis by hypoxia-inducible factors.
      Currently, umbilical cord blood gas pH is generally used as an indicator of fetal status, but it can not be collected under life-threatening conditions such as non-reassuring fetal status. Further, as postnatal blood gas is easily corrected by active resuscitation, it may not be possible to correctly evaluate the hypoxemic condition before or soon after birth. Although the elevation of deviation enzymes such as lactate dehydrogenase (LDH), and creatine kinase (CK) has been widely recognized as useful in identifying damaged tissue,
      • Pincus M.R.
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      Clinical enzymology.
      ,
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      • Maeda H.
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      • et al.
      Brain hypothermia therapy for neonatal hypoxic-ischemic encephalopathy with a severely elevated serum creatine kinase level.
      there are few reports focusing on various blood count indicators related to the severity of fetal distress or neonatal asphyxia. Currently, blood count is frequently used in the diagnosis and follow-up of blood diseases and infectious diseases, even in neonatal infants. If it can be shown that the reticulocyte count soon after birth is related to hypoxemia, it may afford a more useful indicator of hypoxemia. Therefore, we examined the relationships between neonatal reticulocyte count and other perinatal variables.

      2. Materials and methods

      Our study was conducted at the neonatal intensive care unit (NICU) of Ohta Nishinouchi Hospital (ONH) in Fukushima Prefecture, Japan, between April 1, 2016 and July 31, 2019. ONH is a 1086-bed general hospital located in central Fukushima. This study was approved by the Ethics Committee of ONH (Number 42).

      2.1 Definitions

      Gestational age (GA) was determined from the last maternal menstrual period, obstetric history and examination, and prenatal ultrasound findings. Day 0 was defined as the day of birth. Small for GA (SGA) was defined as a BW below the 10th percentile for GA, in accordance with the gender-specific growth charts of Itabashi.
      • Itabashi K.
      • Miura F.
      • Uehara R.
      • Nakamura Y.
      New Japanese neonatal anthropometric charts for gestational age at birth.
      Gestational diabetes mellitus (GDM) was defined as any degree of glucose intolerance using one or more values from a 75-g oral glucose tolerance test.
      • Metzger B.E.
      • Gabbe S.G.
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      • Catalano P.A.
      • et al.
      International Association of Diabetes and Pregnancy Study Groups Consensus Panel
      International association of diabetes and pregnancy study groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy.
      Hypertensive disorder of pregnancy (HDP) was diagnosed after 20 weeks of gestation as cases with blood pressure levels ≧140/90 mmHg on two occasions at least 4h apart, or in those with chronic hypertension.
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      • et al.
      Hypertensive disorders of pregnancy: ISSHP classification, diagnosis, and management recommendations for international practice.
      Magnesium (Mg) sulfate was intravenously administered to women at risk of preterm delivery before 32 weeks of gestation.
      • World Health Organization
      Magnesium sulfate for fetal protection from neurological complications, WHO recommendations on interventions to improve preterm birth outcomes. Geneva.
      Although Mg sulfate is ineffective in delaying birth or preventing preterm birth,
      • Crowther C.A.
      • Brown J.
      • McKinlay C.J.
      • Middleton P.
      Magnesium sulphate for preventing preterm birth in threatened preterm labour.
      the World Health Organization recommends it for the protection of the fetus from neurological complications.
      • World Health Organization
      Magnesium sulfate for fetal protection from neurological complications, WHO recommendations on interventions to improve preterm birth outcomes. Geneva.
      Antenatal corticoid therapy (ACT) was administered to women likely to deliver before 34 weeks of gestation.
      Effect of corticosteroids for fetal maturation on perinatal outcomes. NIH Consensus Development Panel on the Effect of Corticosteroids for Fetal Maturation on Perinatal Outcomes..
      Respiratory distress syndrome was defined as any respiratory symptom (tachypnea, retraction, grunting or cyanosis) observed soon after birth and which was improved by endotracheal surfactant therapy. These definitions were used consistently throughout the study period.

      2.2 Resuscitation

      Neonatal resuscitation was conducted in our hospital in accordance with the Japan Resuscitation Council Guidelines 2015 (based on the 2015 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science with Treatment Recommendations).
      • Hazinski M.F.
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      • et al.
      Part 1: Executive Summary: 2015 International consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations.
      If any unstable conditions (premature babies, weak breathing, or hypo-tonus) were observed soon after birth, routine care, such as warming, positioning, opening of airways, and skin stimulation, was started. We confirmed breathing and heart rate within 1 min after birth. If spontaneous respiration was recognized and the heart rate was 100−/−min or more, we next confirmed the presence of respiratory effort and/or cyanosis. When both were recognized, continuous positive airway pressure (CPAP) or oxygen was administered.
      • SUPPORT Study Group of the Eunice Kennedy Shriver NICHD Neonatal Research Network
      • Finer NN
      • Carlo WA
      • Walsh MC
      • Rich W
      • Gantz MG
      • et al.
      Early CPAP versus surfactant in extremely preterm infant.
      ,
      • O'Donnell C.P.
      • Kamlin C.O.
      • Davis P.G.
      • Carlin J.B.
      • Morley C.J.
      Clinical assessment of infant colour at delivery.
      If dyspnea was not improved, positive pressure ventilation (PPV) by bag and mask were administered. If there was no spontaneous breathing by the neonate or if the heart rate was less than 100−/−min within 1 min after birth, we started PPV by bag and mask.
      • Kattwinkel J.
      • Short J.
      • Shavell L.
      • Siede B.
      Use of resuscitation devices for positive-pressure ventilation.
      When the heart rate did not improve to 100−/−min or more, tracheal intubation was performed after assessing whether ventilation was appropriate. Umbilical cord milking (UCM), consisting of gently grasping the uncut umbilical cord and squeezing the cord from the placenta several times toward the infant, was routinely performed for infants <29 weeks of gestation.
      • Perlman J.M.
      • Wyllie J.
      • Kattwinkel J.
      • Wyckoff M.H.
      • Aziz K.
      • Guinsburg R.
      • et al.
      Part 7: neonatal resuscitation: 2015 International consensus on cardiopulmonary resuscitation and emergency cardiomuscular care science with treatment recommendations.
      In this study, no resuscitation was defined as the achievement of a stable condition soon after birth by routine care only.

      2.3 Measurements

      All blood samples from enrolled infants were collected into EDTA (ethylenediaminetetraacetic acid dipotassium salt dehydrate) laboratory tubes within 3 h after admission to the NICU. The sample volume was 100 μL, and reticulocyte and full blood counts were analyzed using a Sysmex XE-2100 system (Sysmex Ltd. Kobe, Japan). The normal reticulocyte count at birth is unknown. However, as Paterakis
      • Paterakis G.S.
      • Lykopoulou L.
      • Papassotiriou J.
      • Stamulakatou A.
      • Kattamis C.
      • Loukopoulos D.
      Flow-cytometric analysis of reticulocytes in normal cord blood.
      reported that reticulocytes comprise 17‰–50‰ of normal umbilical cord blood, it is presumed to be about the same level. In this study, the median reticulocyte count during the early postnatal period was 60.1‰ (range: 21.9 to 148.6). For this reason, 60‰ was set as a cut-off value, with reticulocyte counts less than 60‰ classified as low, and with those 60‰ or more classified as high in this study.

      2.4 Perinatal variables

      The following perinatal variables were examined: (1) gender, (2) multiple birth (singleton or twin), (3) GA, (4) BW, (5) SGA, (6) umbilical artery pH (UmApH), (7) Apgar 5 min, (8) maternal age, (9) maternal gravidity, history of (10) smoking, (11) alcohol, (12) GDM, (13) HDP, (14) Mg sulfate administration, (15) ACT, (16) delivery type [vaginal or cesarean section (C-section)], (17) maternal Hb before delivery, (18) tracheal intubation, (19) CPAP or Bag and Mask, (20) maximum fraction of inspired oxygen (FiO2) at resuscitation, (21) body temperature (BT) on admission, (22) surfactant therapy, (23) white blood cells (WBCs), (24) Hb, (25) nucleated erythrocytes, (26) hematocrit, (27) platelets, (28) mean platelet volume (MPV), (29) LDH, and (30) CK.

      2.5 Data collection and statistical analyses

      The data shown below were collected from the perinatal records. All univariate analyses were performed using SPSS II for Windows (SPSS Inc., Chicago, Ill.). For categorical variables such as gender, the Mann–Whitney U test was used for comparisons, while continuous variables such as GA were compared using Spearman's rank correlation coefficient, and data are presented as the median and range. In consideration of the sample size, we used 0.05 as the cutoff value to select items to be entered for multivariate analysis. Multivariate logistic regression analysis was performed using SPSS II, with 19 variables related to reticulocyte count further analyzed. To determine their association with reticulocyte count, the odds ratio (OR) and its 95% confidence interval (CI) were calculated. Statistical significance was defined as p < 0.05.

      3. Results

      3.1 Patients

      A total of 810 neonatal infants were admitted to the NICU during the study period. Overall, 382 infants were excluded on the basis of the predefined criteria: (1) admission after 1 postnatal day (n = 300), (2) birth outside our hospital (n = 49), (3) presence of chromosomal abnormalities (n = 5, trisomy21, 4; trisomy18, 1), (4) congenital major malformations (n = 8, anal atresia, 2; achondroplasia, 2; Ebstein's anomaly, 1; giant patent ductus arteriosus, 1; choanal atresia nose, 1; hypoplastic lung, 1), (5) monochorionic diamniotic infants experiencing twin-to-twin transfusion syndrome (n = 4), and (6) insufficient clinical information (n = 16). No infants experienced feto-maternal transfusion syndrome or hemolytic diseases such as blood type incompatibility that could cause fetal anemia. Finally, a total of 428 infants were analyzed (213 in the low reticulocyte count group and 215 in the high group).

      3.2 Clinical characteristics of the 428 enrolled infants and their mothers

      Of the 428 infants, 317 (74.1%) were preterm and 111 (25.9%) were term. Table 1 shows the perinatal characteristics of the 428 infants (and their mothers) included in this study. More male (n = 232) than female (n = 196) infants were enrolled. The median GA was significantly higher in the low group at 37 weeks 0 days (range: 27 week 0 days–41 week 5 days) than in the high group (32 weeks 2 days, range: 24 weeks 1 day–38 weeks 0 days) (p < 0.01). The median BW in the low group was significantly higher than that in the high group (2235 g, range: 744 to 4342 g vs. 1667 g, range: 465 to 3240 g) (p < 0.01). The numbers of SGA infants in the low and high groups were 68 (31.9%) and 39 (18.1%), respectively (p < 0.01). The median UmApH values in the low and high groups were 7.316 (range: 6.529 to 7.511) and 7.349 (range: 6.606 to 7.525), respectively (p < 0.01). The numbers of mothers administered Mg sulfate in the low and high groups were 67 (31.5%) and 116 (54.0%), respectively (p < 0.01). The numbers of mothers receiving ACT in the low and high groups were 39 (18.3%) and 129 (60.0%), respectively (p < 0.01), and the numbers of infants born by C-section in the low and high groups were 134 (62.9%) and 165 (76.7%), respectively (p < 0.01). The median maternal Hb in the low group was significantly higher than that in the high group (11.3 g/dL, range: 6.4–15.5 g/dL vs. 10.7 g/dL, range: 7.7–15.2 g/dL) (p < 0.01). However, there were no significant differences between the two groups with regard to multiple births, maternal age and gravidity, maternal history of smoking and alcohol, DM and hypertension during the pregnancy.
      Table 1Characteristics of the 428 infants and their mothers.
      Grouplow (n = 213)high (n = 215)p value
      Gender, male, n (%)129 (60.6)103 (47.9)0.01
      Multiple births, twin, n (%)37 (17.4)50 (23.3)0.13
      GA, wks, median (range)37wk0d32wk2d<0.01
      (27wk0d−41wk5d)(24wk1d−38wk0d)
      BW, g, median (range)2235 (744−4342)1667 (465−3240)<0.01
      SGA, n (%)68 (31.9)39 (18.1)<0.01
      UmApH, median (range)7.316 (6.529–7.511)7.349 (6.606–7.525)<0.01
      Apgar 5 min, median (range)9 (1–9)8 (2–9)<0.01
      Maternal age, year, median (range)32 (17–45)31 (18–43)0.17
      Gravidity, median (range)0 (0–8)0 (0–6)0.25
      Smoking, n (%)49 (23.0)61 (28.4)0.10
      Alcohol, n (%)42 (19.7)45 (20.9)0.81
      GDM, n (%)16 (7.5)10 (4.7)0.22
      HDP, n (%)43 (20.2)34 (15.8)0.19
      Mg sulfate administration, n (%)67 (31.5)116 (54.0)<0.01
      ACT, n (%)39 (18.3)129 (60.0)<0.01
      Delivery type, C-section, n (%)134 (62.9)165 (76.7)<0.01
      Maternal Hb, g/dL, median (range)11.3 (6.4–15.5)10.7 (7.7–15.2)<0.01
      GA, gestational age; BW, birth weight; SGA, small for gestational age; UmApH, umbilical artery hydrogen ion concentration; GDM, gestational diabetes mellitus; HDP, hypertensive disorder of pregnancy; Mg, magnesium; ACT, antenatal corticosteroid therapy; C-section, cesarean-section; Hb, haemoglobin.

      3.3 Clinical courses

      Table 2 shows the clinical courses of the 428 infants. The number of infants receiving tracheal intubation at resuscitation was significantly lower in the low group than in the high group [11 (5.2%) vs. 72 (33.5%)] (p < 0.01). The median maximum FiO2 values at resuscitation in the low and high groups were 21 (range: 21 to 100) and 40 (range: 21 to 100), respectively (p < 0.01). The median BT values on admission in the low and high groups were 36.6 (range: 34.9 to 38.8) and 36.3 (range: 34.4 to 37.7), respectively (p < 0.01). The number of infants receiving endotracheal surfactant therapy was also significantly lower in the low group than in the high group [20 (9.4%) vs. 92 (42.8%)] (p < 0.01). Four infants died during the study period: one term infant died due to severe birth asphyxia, and 3 extremely low BW infants died due to sepsis.
      Table 2Clinical courses of the 428 infants.
      Grouplow (n = 213)high (n = 215)p value
      Tracheal intubation, n (%)11 (5.2)72 (33.5)<0.01
      CPAP, or Bag and mask, n (%)90 (42.3)93 (43.3)0.83
      Maximum FiO2, %, median (range)21 (21–100)40 (21–100)<0.01
      BT, °C, median (range)36.6 (34.9–38.8)36.3 (34.4–37.7)<0.01
      Surfactant therapy, n (%)20 (9.4)92 (42.8)<0.01
      CPAP, continuous positive airway pressure; FiO2, fraction of inspiratory oxygen; BT, body temperature.

      3.4 Laboratory findings

      The median reticulocyte counts (‰) for all GAs were as follows (Fig. 1): 24–25 wks (n = 11), 124.1 (range: 106.3 to 148.6); 26–27 wks (n = 25), 111.1 (range: 55.5 to 144.3); 28–30 wks (n = 52), 81.9 (range: 35.6 to 128.6); 31–33 wks (n = 86), 71.6 (range: 28.3 to 116.6); 34–36 wks (n = 143); 59.6 (range: 30.2 to 110.9); and 37–41 wks (n = 111), 43.2 (range: 21.9 to 69.2). Table 3 shows the laboratory findings on admission. The median nucleated erythrocyte counts (/100WBC) in the low and high groups were 3 (range: 0 to 164) and 9 (range: 0 to 498), respectively (p < 0.01). The median MPV values in the low and high groups were 9.4 (range: 7.8 to 11.8) and 9.2 (range: 7.6 to 12.1), respectively (p = 0.01). However, there were no significant differences between the two groups with regard to Hb, hematocrit, and platelet.
      Figure 1
      Figure 1Relationships between neonatal reticulocyte count and GA. Higher reticulocyte counts were observed in more preterm infants.
      Table 3Laboratory findings for the 428 infants.
      Grouplow (n = 213)high (n = 215)p value
      WBCs,/μL, median (range)12,3009600<0.01
      (2900−39,500)(4600−30,700)
      Hb, g/dL, median (range)17.9 (12.0–24.3)17.7 (11.8–25.3)0.17
      Nucleated erythrocyte,/100WBC, median (range)3 (0–164)9 (0–498)<0.01
      Hematocrit, %, median (range)52.0 (36.7–67.3)51.2 (34.9–71.9)0.11
      Platelets, × 104/μL, median (range)25.3 (10.2–57.6)24.2 (8.5–47.6)0.05
      MPV, median (range)9.4 (7.8–11.8)9.2 (7.6–12.1)0.01
      LDH, IU/L, median (range)431 (245−2355)408 (165−1248)0.03
      CK, IU/L, median (range)396 (63−2566)280 (32−2360)<0.01
      WBCs, white blood cells; Hb, haemoglobin; MPV, mean platelet volume; LDH, lactate dehydrogenase; CK, creatine kinase.

      3.5 Clinical significance of neonatal reticulocyte count during the early postnatal period

      Table 4 shows the relationships between the reticulocyte count and other perinatal variables as determined by multivariate logistic regression analysis. There were significant relationships between the neonatal reticulocyte count and gender (p < 0.01, OR, 0.37; 95% CI, 0.21 to 0.64), GA (p < 0.01, OR, 0.92; 95% CI, 0.90 to 0.93), delivery type (p = 0.03, OR, 0.51; 95% CI, 0.28 to 0.95), maternal Hb before delivery (p < 0.01, OR, 0.74; 95% CI, 0.60 to 0.91), tracheal intubation at resuscitation (p = 0.04, OR, 2.75; 95% CI, 1.04 to 7.32) and MPV (p < 0.01, OR, 0.51; 95% CI, 0.35 to 0.74). The neonatal reticulocyte count was higher for female than for male infants, and for preterm birth than full-term delivery infants during the early postnatal period. It was also higher in infants born by C-section than in those born by vaginal delivery. In addition, it was higher in infants with tracheal intubation than in those without tracheal intubation at resuscitation. Further, negative correlations were observed between neonatal reticulocyte count and maternal Hb before delivery and MPV.
      Table 4Relationships between RC and other perinatal variables determined by multivariate logistic regression analysis.
      p valueOR95% CI
      Gender<0.010.370.21–0.64
      GA<0.010.920.90–0.93
      Delivery type0.030.510.28–0.95
      Maternal Hb<0.010.740.60–0.91
      Tracheal intubation0.042.751.04–7.32
      MPV<0.010.510.35–0.74
      BW0.71
      SGA0.28
      UmApH0.58
      Apgar 5 min0.52
      Mg sulfate administration0.38
      ACT0.38
      Maximum FiO20.84
      BT0.77
      Surfactant therapy0.98
      WBC0.48
      Nucleated erythrocyte0.52
      LDH0.07
      CK0.97
      GA, gestational age; Hb, haemoglobin; MPV, mean platelet volume; BW, birth weight; SGA, small for gestational age; UmApH, umbilical artery hydrogen ion concentration; Mg, magnesium; ACT, antenatal corticosteroid therapy; FiO2, fraction of inspiratory oxygen; BT, body temperature; WBCs, white blood cells; LDH, lactate dehydrogenase CK, creatine kinase.

      3.6 Sub-analysis for term infants

      A total of 111 term infants were sub-analyzed (107 in the low reticulocyte count group and 4 in the high group). No significant relationships between the neonatal reticulocyte count and perinatal variables were determined by multivariate logistic regression analysis (Data are not shown).

      4. Discussion

      This retrospective cohort study examined the relationships between neonatal reticulocyte count and other perinatal variables. Moreover, we observed significant relationships between neonatal reticulocyte counts and gender, GA, delivery type, maternal Hb before delivery, tracheal intubation at resuscitation and MPV during the early postnatal period.
      Two hundred and ninety-nine (69.9%) of the infants enrolled in this study were born by C-section. Compared with matched controls, infants with persistent pulmonary hypertension are more likely to have been delivered by C-section.
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      Pulmonary vascular transition at birth can be influenced by type of delivery, asphyxia, BT, and oxygen concentration used in the resuscitation gas. However, there were no significant relationships between neonatal reticulocyte count and UmApH, Apgar 5 min, maximum FiO2 at resuscitation or BT on admission in this study. On the other hand, many infants required tracheal intubation at resuscitation in the high group. Neonatal hypoxemic respiratory failure is often caused by surfactant deficiency and ventilation-perfusion mismatch. Our multivariate logistic regression analysis revealed that whether tracheal intubation was required rather than bag and mask ventilation or oxygen concentration had a strong correlation with reticulocyte count.
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      Umbilical cord blood erythroblast count as an index of intrauterine hypoxia.
      reported that nucleated RBC and erythroblast counts were significantly higher in infants born by vaginal delivery than in those born by C-section. The nucleated RBC counts for vaginal delivery were related to the length of labor. However, our study showed that higher reticulocyte counts were associated with C-section. Infants born by vaginal delivery may be released from the hypoxemic conditions associated with labor by birth. On the other hand, infants born by C-section experience a more rapid environmental change from inside to outside the uterus compared with infants born by vaginal delivery. The clinical significance of the physiological response to hypoxemic conditions before and after birth may differ depending on the difference in delivery type.
      Kumar et al.
      • Kumar A.
      • Rai A.K.
      • Basu S.
      • Dash D.
      • Singh J.S.
      Cord blood and breast milk iron status in maternal anemia.
      reported that Hb concentrations were significantly lower in the cord blood of anemic mothers and showed linear relationships with maternal Hb. Moreover, they showed that, even with maternal anemia, the fetal hematological condition remains normal. Similar results were observed in our study; there was no significant difference between the two groups with regard to Hb, despite the significant difference with regard to maternal Hb before delivery. Higher reticulocyte counts in cord blood can be attributed to the active hematological status to the fetus; however, the details remain controversial.
      MPV is recognized an important predictor of cardiovascular risk in adults.
      • Chu S.G.
      • Becker R.C.
      • Berger P.B.
      • Bhatt D.L.
      • Eikelboom J.W.
      • Konkle B.
      • et al.
      Mean platelet volume as a predictor of cardiovascular risk: a systematic review and meta-analysis.
      On the other hand, Gioia et al.
      • Gioia S.
      • Piazze J.
      • Anceschi M.M.
      • Cerekja A.
      • Alberini A.
      • Giancotti A.
      • et al.
      Mean platelet volume: association with adverse neonatal outcome.
      reported that low MPV is associated with more frequent oxygen therapy or need for mechanical ventilation at 48 h of life in preterm infants from mothers with abnormal blood flow velocity in the uterine arteries. Our study showed that higher reticulocyte counts were associated with tracheal intubation and lower MPV, but not with oxygen therapy in neonatal infants.
      One hundred and seven (25.0%) SGA infants were included in this study. SGA is considered to be a multifactorial condition in which both genetic and environmental factors, such as maternal young and advanced age, low maternal body mass index, short stature, mother born as SGA, and cigarette smoking, play a role.
      • Goldenberg R.L.
      • Culhane J.F.
      • Iams J.D.
      • Romero R.
      Epidemiology and causes of preterm birth.
      Pregnant women with complications such as hypertensive disorders and inflammatory responses can lead to placental oxidative stress due to poor placentation and dysfunctional utero-placental perfusion. Moreover, Hasse
      • Haase V.H.
      Regulation of erythropoiesis by hypoxia-inducible factors.
      reported that the physiological response to systemic hypoxemia involves an increase in RBC production through the induction of increased erythropoietin production in the kidney. Based on our results, the increase in nucleated erythrocyte and reticulocyte counts during the early postnatal period was constant even in more premature births. Interestingly, there was no significant relationship between neonatal reticulocyte count and SGA. Although SGA is thought to be a multifactorial chronic condition, SGA infants may demonstrate differences in their responsiveness to erythropoietin against acute conditions such as postnatal respiratory distress. Moreover, there was a significant relationship between neonatal reticulocyte count and gender. Higher reticulocyte counts were observed in female preterm infants. This unexpected result may reflect a greater vulnerability to stress, such as delivery type, in male preterm infants and warrants further investigation.
      This study has several limitations. First, since only NICU infants were examined, it is biased as a study group in the sense that healthy infants were not included in this study. Second, the reticulocyte count is considered to be increased with earlier GA. Bizzarro and colleagues
      • Bizzarro M.J.
      • Colson E.
      • Ehrenkranz R.A.
      Differential diagnosis and management of anemia in the newborn.
      reported that the reticulocyte counts in the fetal period are 96 ± 32‰ at 26 weeks of gestation and thereafter decrease as the fetus approaches maturity, reaching 32 ± 14‰ at 37–41 weeks of gestation. Although our results also showed a similar tendency, the measured reticulocyte counts were larger than those in the previous report. Since many premature infants present with respiratory distress soon after birth, additional analysis was performed only for term infants to exclude prematurity. However, there were no significant relationships between the neonatal reticulocyte count and perinatal variables. This cut-off value was considered to be inappropriate for additional analysis because few infants were classified into the high group. Third, the routine use of UCM in <29 weeks-gestation infants is recommended
      • Perlman J.M.
      • Wyllie J.
      • Kattwinkel J.
      • Wyckoff M.H.
      • Aziz K.
      • Guinsburg R.
      • et al.
      Part 7: neonatal resuscitation: 2015 International consensus on cardiopulmonary resuscitation and emergency cardiomuscular care science with treatment recommendations.
      ; however, the long-term safety profile remains controversial. Katheria
      • Katheria A.
      • Blank D.
      • Rich W.
      • Finer N.
      Umbilical cord milking improves transition in premature infants at birth.
      reported that infants undergoing cord milking demonstrated an increased heart rate and oxygen saturation within the first 5 min of birth compared to those receiving immediate cord clamping. The exact physiological impact of cord milking on neonatal adaptation requires further clarification. Last, we did not directly evaluate hypoxemia by measurement of erythropoietin in the cord blood. We simply performed a substitute evaluation by respiratory support management at resuscitation. There are a number of clinical indicators, such as arterial oxygen saturation, partial pressure of arterial oxygen/FiO2 and alveolar-arterial oxygen difference, for evaluating hypoxemia at resuscitation. However, continuous monitoring using arterial blood gas analysis has not been conducted in either case.
      In conclusion, we observed significant relationships between neonatal reticulocyte count and gender, GA, delivery type, maternal Hb before delivery, tracheal intubation at resuscitation and MPV. A higher neonatal reticulocyte count in NICU infants may be one of the physiological responses to a more rapid environmental change during the early postnatal period. Further studies on larger populations are necessary to evaluate the clinical significance of neonatal reticulocyte count during the early postnatal period.

      Author's specific contributions

      All authors have participated in clinical study design and interpretation. MY drafted the first version of the manuscript. TI and AG analyzed the data of the study. MY, MC and YK collected the data of the study (including medical treatment) and revised a part of the manuscript critically. TI and AG reviewed the manuscript critically. All authors read and approved the final manuscript.

      Declaration of Competing Interest

      All authors have no conflict of interest to declare in relation to this study.

      Acknowledgement

      The authors are grateful to the members of the NICU and clinical laboratory room at Ohta General Hospital Foundation Ohta Nishinouchi Hospital, Fukushima, Japan.

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