ucresubjan25.1 1 Reference values for umbilical cord diameters in 2 placenta specimens 3 H. Pinar1, Murat Iyigün2 4 5 Halit Pinar, MD 6 Brown Medical School 7 Women and Infants Hospital 8 Division of Perinatal Pathology 9 101 Dudley Street 10 Providence, Rhode Island 02905 11 12 Murat Iyigün, PhD 13 University of Colorado 14 Department of Economics 15 Boulder, CO 16 17 Corresponding Author: 18 Halit Pinar, MD 1 ucresubjan25.1 19 Division of Perinatal and Pediatric Pathology 20 Brown Medical School-Women and Infants Hospital 21 101 Dudley Street 22 Providence, RI, 02905 23 Phone: (401) 274-1122 1190 24 Fax: (401) 453-7681 25 hpinar@wihri.org 26 Running Title: reference values for umbilical cord diameters in placenta 27 specimens 2 ucresubjan25.1 28 Abstract 29 Context 30 To determine the normal values for umbilical cord diameters in placenta 31 specimens. 32 Methods 33 We retrospectively collected values of umbilical cord diameters from 973 34 placenta specimens examined in the Division of Perinatal Pathology at Brown 35 University. The specimens were examined using the same standard protocol 36 during the calendar years 2005 t0 2007. Gestational ages of the newborns ranged 37 from 20-41 weeks. Placentas originated from pregnancies associated with any 38 condition known to cause fetal growth impairment were excluded. In addition, 39 cases without complete clinical or pathological information and samples from 40 multifetal gestation, or with developmental abnormalities such as umbilical cord 41 masses were also excluded. The longest diameter of umbilical cords 42 representative of the entire sample was measured and recorded. Since only cord 43 segment(s) close to the placental insertion site were received in the laboratory, the 44 measurements were obtained from these samples. No measurements were 45 available from the remainder of the cords left with the newborns. To establish the 3 ucresubjan25.1 46 link between umbilical cord diameters and gestational age, polynomial regression 47 analysis was conducted. 48 Results 49 Measurements from 973 singleton placentas were used in the statistical 50 estimates and distribution of normal values throughout gestation was calculated. 51 The regression equation was y = - 11.9994 + 1.33966x - .018231x2 where y 52 denotes the umbilical cord diameter in millimeters and x is gestational age in 53 weeks. A statistically significant non-linear relationship was found between 54 umbilical cord diameters and gestational age. The direct measurements of 55 umbilical cords during the pathological examination were smaller than the 56 ultrasound measurements by 30-40% over the gestational age range of 20 to 41 57 weeks. 58 Conclusions 59 Reference values for umbilical cord diameters in placenta specimens were 60 determined and their distribution according to gestational ages was calculated. 61 Our nomograms are novel and derived from the pathological specimens rather 62 than in vivo ultrasound examinations. Therefore, instead of published reference 63 values of umbilical cord thickness in the pathology literature, which reflect in vivo 4 ucresubjan25.1 64 ultrasound measurements, we recommend that the values collected from placental 65 specimens should be used. 5 ucresubjan25.1 66 Introduction 67 Ultrasonographic evaluation of the fetus is very significant in the obstetrical 68 management. In addition to fetal parameters it includes placental measures such 69 as umbilical cord length, diameter, and degree coiling among others [1-8]. The 70 values obtained in the ultrasonographic evaluation are compared with normal 71 reference values allowing certain diagnoses. For example thin umbilical cords 72 have been associated with fetal growth impairment. A study comparing growth- 73 restricted fetuses with an appropriate for gestational age group has shown that the 74 cross-sectional area of all components of the umbilical cord is reduced in the 75 former [9-10]. In pregnancies complicated by early pre-eclampsia, the cross- 76 sectional areas of the Wharton jelly and umbilical vein were found to be reduced 77 in comparison to normal pregnancies [11-13]. An increase in the umbilical cord 78 diameter has been described in pregnancies complicated by gestational diabetes, 79 various causes of macrosomia and aneuploidies [7, 14-15]. 80 Evaluation of the umbilical cord is also an essential part of the pathological 81 examination of the placenta. Proper macroscopic examination technique includes 82 measurement of the length and diameter of the umbilical cord among other 83 features [16-21]. Although most of the normal reference values for placental 6 ucresubjan25.1 84 parameters were derived from pathological samples, umbilical cord diameter was 85 not [19-21]. They have been derived from various studies that used 86 ultrasonography in ongoing pregnancies [18-21]. Umbilical cords in vivo are 87 active vascular conduits connecting the placenta to the fetus and 88 hemodynamically active. Measurements obtained in vivo reflect active blood flow 89 in the umbilical vessels. Blood flow keeps the vessels patent and prevents them 90 from collapse. After delivery, blood flow ceases and the vessels constrict and 91 collapse. These changes affect the shape and dimensions of the umbilical cord. 92 The purpose of this study was to define the reference values of umbilical cord 93 diameters between 20 and 41 weeks gestational ages using placental samples. 94 Materials and method 95 Population 96 We retrospectively reviewed 5,499 placenta specimens that were examined 97 during the years 2005-2006 using the same standard protocol by the Division of 98 Perinatal Pathology at Brown University. Medical records were reviewed for 99 demographic characteristics, maternal antepartum history and associated 100 complications, birth data and neonatal findings. The pathology records of the 101 placenta specimens were also examined and data extracted. 7 ucresubjan25.1 102 Inclusion and exclusion criteria 103 The criteria for placental examination at Women and Infants Hospital include 104 similar conditions to the College of American Pathologists (CAP) 105 recommendations (Table 1) [17]. Since our objective was to collect a cohort as 106 normal as possible, any sample from a mother with any condition that has been 107 associated with fetal or placental growth impairment has been excluded from the 108 study. In addition, cases without complete clinical or pathological information and 109 samples from multifetal gestation, or with developmental abnormalities such as 110 umbilical cord masses were also excluded. Since placentas from term and 111 uncomplicated pregnancies routinely were not sent to the Laboratory to be 112 examined, 503 term placentas were randomly chosen and records reviewed. 372 113 of these placentas were included in the study. 114 Macroscopic examination of the placenta 115 Placentas were examined fresh using a standard method. After inspection, the 116 umbilical cords were trimmed leaving a stump at the insertion site measuring 0.5- 117 1 cm. Next, the membranes were examined and trimmed. The trimmed placental 118 disc was weighed after draining all the blood. The longest and shortest diameters 119 of the placental disc were measured. After the placental disc was sliced into 1 cm 120 thick slices, thickest and thinnest slices were measured. 8 ucresubjan25.1 121 Measuring and sampling of the umbilical cords 122 The length of all the received cord segments was measured. The umbilical 123 cord segment closer to the insertion site to the placental disc was designated as the 124 placental end (proximal end). The opposite segment towards the fetus was 125 designated as the fetal end (distal end). Only the segments of the umbilical cords 126 close to the placental insertion site (proximal) were available for examination. 127 Irrespective of the shape of the cross section, the longest diameter representative 128 of the entire umbilical cord sample was measured and recorded (Fig. 1). 129 Data Analysis 130 Data were collected into a central database and analyzed using Stata Version 131 9.2 (2006, StataCorp LP, College Station, TX). We derived our baseline estimates 132 using a non-linear (polynomial) Ordinary Least Square regression (OLS). We 133 included gestational age as the main explanatory variable (x) and umbilical cord 134 diameter measures as the dependent variable (y). We allowed gestational age to 135 enter the empirical specifications non-linearly (i.e., we included x2 as a separate 136 explanatory variable). This is done to capture a potentially non-linear relationship 137 between gestational age and umbilical cord diameter, whereby the growth rate in 138 the umbilical diameter can vary over gestational time. The distribution of the data 139 by gestational age is summarized in Table 2. As shown, the average umbilical 9 ucresubjan25.1 140 cord diameters peak during the 37th week and then drop somewhat in the 141 remaining four weeks. 142 Results 143 During the study period, a total of 5, 499 placenta specimens were examined 144 between 20 and 41 weeks of gestation. 973 (17.7%) specimens met the inclusion 145 criteria. Maternal ages ranged from 18-49 years, whereas the median parity was 146 two, ranging from zero to five. In all the 973 cases included in the study, 147 estimated date of confinement was determined based on an accurate last 148 menstrual period and confirmed by a first- or second-trimester sonogram. The 149 gestational ages ranged between 20 and 41 weeks. 150 Regressing the umbilical cord diameter (y) on gestational age (x) in a non- 151 linear polynomial OLS equation produced y = - 11.889 + 1.352x - .01888x2 and a 152 fit measure of R2 = 0.172. All variables yielded statistical significance with the 153 intercept term -11.889 yielding P < 0.0001, the coefficient 1.352 on gestation age 154 (x) producing P < 0.0001 and the coefficient of -0.01888 on the squared value of 155 gestational age (x2) generating P < 0.002. The 5th, 95th percentile bands as well as 156 the fitted mean values of umbilical cord measures that the regression equation 157 produced are depicted in Figure 3. 10 ucresubjan25.1 158 Discussion 159 The umbilical cord serves an essential role in fetal intrauterine survival, but 160 for a long period of time, it was one of the least studied components of the fetal 161 anatomy during an ultrasound examination [1-2]. Prenatal morphological 162 assessment of the umbilical cord is usually limited to the evaluation of the number 163 of umbilical cord vessels. Other morphometric umbilical cord parameters, such as 164 cord thickness and the amount of Wharton's substance or umbilical cord coiling 165 have been reported but not routinely used [11-12, 22]. 166 Weissman and colleagues conducted the first study constructing nomograms 167 for the umbilical cord components using ultrasound [2]. The authors established 168 reference measures for the diameters of the umbilical cord, vein, and arteries. In a 169 more recent study Raio et al. published nomograms of the umbilical cord diameter 170 and area according to gestational age from 10 to 42 weeks of gestation [1]. In 171 their study, umbilical cords were evaluated at the level of the umbilical cord 172 insertion on 557 patients. They demonstrated an increase in umbilical cord 173 thickness as a function of gestational age up to 34 weeks of gestation, followed by 174 a reduction of this parameter. These findings were similar to the nomogram 175 published by Weissman except the cessation of cord thickness in the latter study 176 was observed later after 36 weeks [2]. 11 ucresubjan25.1 177 We replicated the findings of Raio et al., which are shown in Figure 4 [1]. 178 Based on a sample of 557 patients and gestational age range of 10 weeks to 41 179 weeks, the Raio et al. study generated y = - 10.0563 + 1.4265x - .0194x2 with 180 umbilical cord diameter as the dependent variable, y, and gestational age and its 181 square as the explanatory variables (x and x2). In Figure 5, we compare Raio, et 182 al.’s findings with ours. As shown, there is considerable similarity between the 183 two results generated with the in vitro direct measurement of the umbilical cord 184 and in vivo measurements obtained by ultrasonography which form the basis of 185 the Raio et al. data [1]. We found a statistically significant non-linear relationship 186 between umbilical cord diameters and gestational age. But the direct 187 measurements of umbilical cords during the pathological examination were 188 smaller than the ultrasound measurements by 30-40% over the gestational age 189 range of 20 to 41 weeks. 190 Although evaluation of the umbilical cord has been part of every 191 recommendation on pathological examination of the placental specimens, 192 reference values for umbilical cord diameters applicable to pathological 193 specimens are not currently available. The sources of the nomograms in the 194 published pathology literature are from ultrasound studies obtained in ongoing 195 pregnancies. Since after delivery the fetal circulation ceases through the umbilical 12 ucresubjan25.1 196 cord, the shape and measurements of this conduit changes. Thus they are not 197 compatible [17-21]. 198 In this study, we determined the reference values for average umbilical cord 199 diameters in placenta specimens and their distribution according to gestational 200 ages. Our nomogram is the first derived from the placental specimens rather than 201 in vivo ultrasound examinations. Since the published reference values of umbilical 202 cord thickness in the pathology literature reflect in vivo ultrasound measurements, 203 they are not appropriate. We recommend the use of the new values during the 204 pathologic examinations. 13 ucresubjan25.1 205 References 206 207 1. Raio L, Ghezzi F, Di Naro E, Gomez R, Franchi M, Mazor M and Brühwiler 208 H. Sonographic measurement of the umbilical cord and fetal anthropometric 209 parameters. Eur J Obstet Gynecol Reprod Biol. 1999;83(2):131-5. 210 2. Weissman A, Jakobi P, Bronshtein M, Goldstein I. Sonographic 211 measurements of the umbilical cord and vessels during normal pregnancies. 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J Clin Pathol. 2008;61(12):1276-84. 17 ucresubjan25.1 269 270 Table 1 Indications of placental examination at Women & Infants Hospital of Rhode Island Placental indications Macroscopic abnormality of the placenta, membranes, or cord noted by U/S or at the delivery Abruptio placenta Retained placenta Suspected small or large placenta Suspected short or long cord (indicate on requisition length of cord that we will not receive) Maternal indications Systemic disorders with clinical concerns for mother or infant Diabetes during any portion of pregnancy Hypertensive disorders Autoimmune disorders Hematologic disorders Seizures Premature delivery Delivery at ≥42 weeks Oligohydramnios or polyhydramnios Peripartum fever and/or infection Clinical concern for infection during gestation – Viruses, including HIV – Bacteria, including Mycobacteria – Fungi – Parasites, etc. Prolonged (≥18 hrs) and/or premature rupture of membranes Heavy or repetitive bleeding other than minor first trimester spotting Abruption Intrauterine invasive procedures with suspected placental, umbilical cord or fetal injury Current known substance abuse or positive drug screen Severe trauma FETAL/NEONATAL INDICATIONS Fetal or perinatal death Fetal or neonatal congenital anomalies, known or suspected Compromised clinical conditions similar but not limited to the following examples: Cord blood ph <7.0 Apgar scores <6 at 5 minutes Ventilatory assistance >10 minutes Anemia - Hct <35% Hydrops fetalis Seizures, persistent hypotonia, and hypoxic-ischemic encephalopathy Infections, known or suspected Intrauterine growth retardation or macrosomia (>4500 g for term infants) Multiple gestation, including vanishing twin 18 ucresubjan25.1 Prematurity 34 weeks or postmaturity weeks Hematologic disorders as defined by: – Anemia of any cause – Erythroblastosis of any cause – Hemoglobinopathies – Thrombocytopenia of any cause 271 272 19