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Published in final edited form as: Am J Obstet Gynecol. 2018 May 5;219(2):187.e1–187.e20. doi: 10.1016/j.ajog.2018.04.047

Femur-Sparing Pattern of Abnormal Fetal Growth in Pregnant Women from New York City After Maternal Zika Virus Infection

Christie L WALKER 1, Audrey A MERRIAM 2, Eric O OHUMA 3, Manjiri K DIGHE 4, Michael GALE Jr 5, Lakshmi RAJAGOPAL 6, Aris T PAPAGEORGHIOU 7, Cynthia GYAMFI-BANNERMAN 8, Kristina M ADAMS WALDORF 9
PMCID: PMC6066422  NIHMSID: NIHMS968041  PMID: 29738748

Abstract

Background

Zika virus (ZIKV) is a mosquito-transmitted flavivirus, which can induce fetal brain injury and growth restriction following maternal infection during pregnancy. Prenatal diagnosis of ZIKV-associated fetal injury in the absence of microcephaly is challenging due to an incomplete understanding of how maternal ZIKV infection affects fetal growth and the use of different sonographic reference standards around the world. We hypothesized that skeletal growth is unaffected by ZIKV infection and that the femur length can represent an internal standard to detect growth deceleration of the fetal head and/or abdomen by ultrasound.

Objective

To determine if maternal ZIKV infection is associated with a femur-sparing pattern of intrauterine growth restriction (IUGR) through analysis of fetal biometric measures and/or body ratios using the INTERGROWTH-21st Project (IG-21) and World Health Organization Fetal Growth Chart (WHO-FGC) sonographic references.

Study Design

Pregnant women diagnosed with a possible recent ZIKV infection at Columbia University Medical Center after traveling to an endemic area were retrospectively identified and included if a fetal ultrasound was performed. Data was collected regarding ZIKV testing, fetal biometry, pregnancy and neonatal outcomes. The IG-21 and WHO-FGC sonographic standards were applied to obtain Z-scores and/or percentiles for fetal head, abdominal circumference (HC, AC) and femur length (FL) specific for each gestational week. A novel IG-21 standard was also developed to generate Z-scores for fetal body ratios with respect to femur length (HC:FL, AC:FL). Data was then grouped within clinically relevant gestational age strata (<24 weeks, 24–27 6/7, 28–33 6/7, >34 weeks) to analyze time-dependent effects of ZIKV infection on fetal size. Statistical analysis was performed using Wilcoxon signed-rank test on paired data, comparing either AC or HC to FL.

Results

A total of 56 pregnant women were included in the study with laboratory evidence of a confirmed or possible recent ZIKV infection. Based on the CDC definition for microcephaly after congenital ZIKV exposure, microcephaly was diagnosed in 5% (3/56) by both the IG-21 and WHO-FGC standards (HC Z-score ≤ −2 or ≤ 2.3%). Using IG-21, IUGR was diagnosed in 18% of pregnancies (10/56; AC Z-score ≤−1.3, <10%). Analysis of fetal size using the last ultrasound scan for all subjects revealed a significantly abnormal skewing of fetal biometrics with a smaller AC versus FL by either IG-21 or WHO-FGC (p<0.001 for both). A difference in distribution of fetal AC compared to FL was first apparent in the 24–27 6/7 week strata (IG-21, p=0.002; WHO-FGC, p=0.001). A significantly smaller HC compared to FL was also observed by IG-21 as early as the 28–33 6/7 week strata (IG-21, p=0.007). Overall, a femur-sparing pattern of growth restriction was detected in 52% of pregnancies with either an HC:FL or AC:FL fetal body ratio less than the 10th percentile (IG-21 Z-score ≤−1.3).

Conclusions

An unusual femur-sparing pattern of fetal growth restriction was detected in the majority of fetuses with congenital ZIKV exposure. Fetal body ratios may represent a more sensitive ultrasound biomarker to detect viral injury in nonmicrocephalic fetuses that could impart long-term risk for complications of congenital ZIKV infection.

Keywords: Biomarker, biometry, biparietal diameter, congenital Zika virus syndrome, femur length, fetal growth restriction, fetus, fetal infection, head circumference, Intergrowth-21, intrauterine growth restriction, IUGR, microcephaly, pregnancy, teratogenesis, ultrasound, virus, Zika

Introduction

Zika virus (ZIKV) is a mosquito-transmitted flavivirus, recently linked to microcephaly following a maternal infection during pregnancy.[1] Vertical transmission of ZIKV has been associated with fetal microcephaly and development of the Congenital ZIKV Syndrome, a condition encompassing a spectrum of fetal neurologic injury including cortical malformations, ventriculomegaly, ocular injury and arthrogryposis.[2, 3, 4] A maternal ZIKV infection has been associated with a rate of birth defects between 5–8%, but may be as high as 13% when infection occurs in the first trimester.[5, 6] Recently, reports of children with a normal head circumference (HC) at birth that were later found to have abnormal brain imaging, ocular injury and postnatal development of microcephaly, has led to the concept that microcephaly does not capture the broader spectrum of ZIKV-associated brain injury.[3, 7, 8, 9, 10] Identification of fetuses with a normal head size that are at risk for long-term adverse outcomes remains limited due to the incomplete knowledge of how a less overt spectrum of ZIKV-associated fetal injury may be detected prenatally. This limitation is further compounded by weaknesses related to diagnostic testing including: 1) inadequate availability of ZIKV testing in regions at risk, 2) lower sensitivity of real-time polymerase chain reaction testing (RT-PCR) due to the transient nature of ZIKV viremia, and 3) lower positive predictive value of serologic testing due to cross-reactivity between ZIKV and related flaviviruses.

In a nonhuman primate model, ZIKV-associated fetal brain injury was associated with an unusual femur-sparing profile of intrauterine growth restriction (IUGR) notable for a growth arrest in ultrasound biometric measures of the fetal head (biparietal diameter, BPD) and abdomen (abdominal circumference, AC) with continued growth of the femur (femur length, FL).[11, 12] This profile of IUGR has been noted as “femur-sparing”[13], but has not been characterized in a clinical study nor is it part of the mainstream categories for IUGR; typically, IUGR has been defined as asymmetric (conserved head growth with lagging growth of the abdomen) or symmetric (equal growth restriction of the head, abdomen and femur).[14]

There is a paucity of data to link aberrant fetal growth in the context of a maternal ZIKV infection to long-term adverse outcomes in the neonate, but IUGR may represent a sensitive indicator of viral injury to the placenta or fetus itself. Whether fetuses exposed to Zika virus with abnormal growth patterns, without microcephaly, may be more susceptible to eye injury or late-onset microcephaly is unknown and represents an important knowledge gap.[15] Although IUGR has been reported in pregnant women with a possible ZIKV infection, the profile of IUGR has not been described.[10, 16] Our objective was to determine if maternal ZIKV infection was associated with a femur-sparing profile of growth restriction, similar to observations in a nonhuman primate model of congenital ZIKV infection.[11, 12] Such an observation may be a first step in identifying nonmicrocephalic fetuses at risk for long-term morbidity.

Materials and Methods

Study Population and Ethics Statement

All pregnant women presenting to Columbia University Medical Center from January 1, 2016 through February 1, 2017 from an area with known ZIKV local transmission were offered screening per Centers of Disease Control (CDC) recommendations. The Columbia University Institutional Review Board approved the study (IRB-AAAQ9686) as a retrospective chart review and informed consent was not required. Cases were excluded if no ultrasound for fetal size or anatomy was completed prior to delivery. The gestational age and due date were estimated according to methods recommended by the American College of Obstetricians and Gynecologists.[17] Following ZIKV diagnosis, a pregnancy ultrasound was performed, and repeated every 3–4 weeks, for the duration of the pregnancy. Timing of ZIKV exposure was estimated based on maternal travel history, but could have occurred later in pregnancy due to sexual exposure from an infected partner; therefore, we included 4 subjects with immediate pre-conception exposure (Table S4). Neonatal outcomes were assessed through measurement of a postnatal HC and head ultrasound scan in the first week of life. A more comprehensive assessment of outcomes was not possible due to limitations on our institutional human subject’s approval and the challenge of data procurement from multiple private pediatric clinics in New York City; therefore, results for some recommended neonatal screening tests were not obtained.

ZIKV Diagnosis

Based on uncertainties in the diagnostic testing for ZIKV infection, we followed CDC convention to describe women as having a “possible” ZIKV infection based on: 1) ZIKV infection detected by ribonucleic acid (RNA) testing on maternal, placental or fetal specimen, or 2) diagnosis of ZIKV infection or unspecified flavivirus infection, timing of infection cannot be determined (i.e., positive/equivocal ZIKV IgM and ZIKV plaque reduction neutralization test (PRNT) titer ≥ 10, regardless of dengue virus PRNT value; or negative ZIKV IgM, and positive or equivocal dengue virus IgM, and ZIKV PRNT titer ≥ 10, regardless of dengue virus PRNT titer).[18, 19] We also followed CDC guidance for the interpretation of laboratory testing of the infant for evidence of congenital ZIKV infection.[18] Any positive nucleic acid test from a serum, urine or cerebrospinal fluid sample was considered a confirmed congenital ZIKV infection. Any non-negative IgM result (e.g. positive, equivocal) from infant serum with a negative nucleic acid test was considered a probable congenital ZIKV infection.

Ultrasound Methodology

The INTERGROWTH-21st (IG-21) sonographic standard was used to derive Z-scores for HC, AC and FL, as well as ratios for HC:FL and AC:FL.[20, 21, 22] Ultrasound scans were originally performed using Hadlock methodology, which measures BPD in a cross-section view from outer-to-inner skull edges. As IG-21 measures the BPD from outer-to-outer skull edges, BPD measurements in this study were not directly translatable to the IG-21 sonographic standard. We chose instead to focus the analysis on HC, AC and FL measurements from which we could directly calculate Z-scores. As the sonographic standard or reference used to interpret fetal size is expected to influence detection of IUGR in pregnancies with maternal ZIKV infection, we also corroborated the findings by applying references from the WHO sponsored Fetal Growth Chart study (WHO-FGC).[20, 23]

Online calculators were used to obtain Z-scores for IG-21[22] and published charts allowed estimation of percentiles for WHO-FGC.[20, 23] Notably, the WHO recommends that diagnosis of ZIKV-associated microcephaly use the IG-21 standard when the gestational age is accurately known and WHO-FGC when gestational age is not reliably known.[24] Studies of pregnancy outcomes from Brazilian women with ZIKV infection have also used the IG-21 standard to determine distribution of fetal biometric measures.[9, 25, 26]

We did not evaluate our data based on sonographic standards developed in the U.S. for two reasons. First, the 1983 Hadlock standard (N=392) was based on a relatively small cohort of Caucasian women and has anecdotally been associated with a common diagnosis of “short femur”.[27, 28, 29, 30] Second, application of racial/ethnic specific standards based on the NICHD Fetal Growth Study (N=2,334)[31] would only have allowed for assignment of biometric measures within ranges of centiles (i.e.. <3rd, 3rd – 5th, 5th–10th), but not a more precise and quantitative analysis necessary to test our hypothesis. Our data on subject ethnicity was also incomplete. We ultimately chose to compare our data to the IG-21 and WHO-FGC standards as they were large population-based studies from multiple countries that included an ethnically diverse cohort. Notably, we could also use the IG-21 standard to specifically test our hypothesis of a femur-sparing profile of fetal growth restriction using fetal body ratios.

Definitions for Microcephaly and IUGR

Variations in the definition for prenatal diagnosis of microcephaly with possible ZIKV infection exist among guidelines and standards.[10, 32, 33] The International Society for Ultrasound in Obstetrics & Gynecology recommends heightened surveillance with specialist referral and neurosonography for fetuses with a HC smaller than 2 standard deviations below the mean (Z-score ≤ −2 SD).[34] The WHO definition for fetal microcephaly, in the context of ZIKV infection, is a HC ≤ −2 SD below the mean.[33] After birth, the CDC definition for microcephaly is a HC less than the 3rd centile for gestational age in the setting of congenital ZIKV exposure (≤ −2 SD).[35] Based on this guidance, we defined microcephaly in our study as a fetal HC Z score ≤ −2 (2.3%, IG-21) or less than the 3rd centile (WHO-FGC).

There is no gold standard to define IUGR and it has been variably defined by deviation of fetal size from a normal distribution at either the 10th, 5th or 3rd centile.[36, 37] The estimated fetal weight (EFW) and AC are consistently identified as important parameters in making the diagnosis and a typical threshold is less than the 10th centile; however, this definition will include many constitutionally small fetuses and miss growth restricted fetuses that are larger than the 10th centile.[38] In this study, we present results using both a conservative (AC <3%, ~Z score ≤ −2) and traditional (AC <10%, ~Z score ≤ −1.3) definition for IUGR to allow comparison of results with AC:FL, a fetal body ratio for AC normalized to FL. Due to the difference in BPD measurements between Hadlock and IG-21, BPD could not be used to calculate EFW; therefore, EFW was not used as a measure of IUGR in this study.

Estimating Population Distribution of Fetal Body Ratios

Fetal body ratios normalized to FL were hypothesized to represent a more sensitive method to detect aberrant growth patterns in fetuses with congenital ZIKV exposure. This approach has the advantage of directly addressing our hypothesis by comparing the size of fetal structures (i.e. head, abdomen) to FL for each fetus, but may not detect constitutionally small fetuses and fetuses with symmetric IUGR. The WHO-FGC has published ratios for FL:HC, but values often overlapped several strata making it difficult to categorize some cases into discrete strata.[20] Therefore, we focused attention on the IG-21 standard from which we could calculate Z-scores for HC:FL and AC:FL.

Published thresholds for IG-21 body ratios did not exist; therefore, we developed these formulas, including mean and standard deviations from the original data (means and standard deviations by gestational week shown in Tables S1, S2, S3). Statistical methods used to construct the fetal biometry ratios were selected using a previously published strategy.[21, 39] In brief, fractional polynomial regression was used, and the resulting functional form further modelled in a multi-level framework to account for the longitudinal design of the study. Goodness-of-fit was evaluated with visual inspection of overall model fit using quantile-quantile plots of the residuals, plots of residual versus fitted values and the distribution of fitted Z-scores across gestational age. All models and goodness-of-fit assessments were fitted with STATA, version 11.2, software (StataCorp LP, College Station, Texas, USA).

Statistical Analysis

Raw measurements for all biometric measures were recorded in millimeters (mm). We analyzed the data in clinically relevant gestational age strata for two reasons: 1) identifying a gestational age threshold at which ZIKV-associated abnormal fetal growth is typically observed has clinical relevance and 2) the effects of ZIKV infection on fetal growth are likely time-dependent with more significant effects occurring in later pregnancy. Gestational age strata were chosen to correspond to transitions classically associated with neonatal viability (18–24 weeks) and morbidity (late second trimester: 24–28 weeks, early third trimester: 28–34 weeks, and near term ≥ 34 weeks). The latest ultrasound per subject was analyzed in each gestational age strata. Wilcoxon signed rank test was used to compare distribution of paired Z-scores for HC to FL or AC to FL. Statistical significance was reported for p values <0.05. Analysis was completed using STATA version 11.2, software (StataCorp LP, College Station, Texas, USA).

Results

ZIKV Diagnosis and Timing of Exposure

Study participants were pregnant women diagnosed with ZIKV infection after travel to countries with local transmission, who received obstetrical care from Columbia University Medical Center (New York City, NY, USA) between January 1, 2016 and February 1, 2017. A total of 66 pregnant women were retrospectively identified with a recent ZIKV infection and 56 were included based on availability of ultrasound data within the Columbia University health care system. The cohort was of mixed race/ethnicity: 12 Hispanic/White, 7 Hispanic/Black, 2 Hispanic/Pacific Islander, 3 White, and 32 other (unknown/more than one race). Thirteen women (13/56, 23%) recalled symptoms consistent with ZIKV infection including a rash, conjunctivitis, fever and myalgias (Table S4). ZIKV infection was diagnosed based on laboratory evidence for a confirmed ZIKV infection (N=21) or unspecified flavivirus infection (N=35; Table S4) according to the U.S. Zika Pregnancy Registry criteria.[5, 40] By travel history, ZIKV exposure was estimated to have occurred immediately preconception (N=4) or in the first (N=16) or second trimester (N=11). An additional 25 women were more uncertain of exposure timing due to prolonged stays in endemic areas and presented to care in the late second or third trimester (mean 30.8 ± 4.5 weeks).

Pregnancy and Birth Outcomes

Prenatal ultrasound was performed between 14 and 40 weeks gestation with each subject typically having 3 ultrasound scans [range 1–7; ≥3 scans, N=29 (52%); 2 scans, N= 15 (27%); 1 ultrasound, N=12 (21%)]. During pregnancy, microcephaly was diagnosed in 5% (3/56) of fetuses by both the IG-21 (HC Z-score ≤ −2) and WHO-FGC (≤ 3rd centile; Table S5). Apart from isolated choroid plexus cysts, no other intracranial abnormalities were detected on prenatal ultrasound. IUGR was diagnosed in 18% of pregnancies by a traditional definition (10/56; AC Z-score ≤−1.3, <10th centile) and 9% by a conservative definition (5/56; AC Z-score ≤−2 or ≤ 2.3 centile, Table 1) using IG-21 standards. The mean Z-score for birthweight for the entire cohort was 0.2 ± 1.0.

Table 1.

Rates of Microcephaly and IUGR by Exposure Time

Exposure Time Gestational Age at Delivery (weeks) Prenatal Diagnosis of Microcephaly (HC <3%) Prenatal Diagnosis of IUGR Birthweight* (g) Birthweight (% IG-21)
WHO-FGC IG-21 AC <3% AC <10%
WHO-FGC IG-21 WHO-FGC IG-21
All (N=56) 37.4 (4.2) 3 (5) 3 (5) 5 (9) 5 (9) 8 (14) 10 (18) 3159 (659) 55 (28.9)
Preconception (N=4) 38 (0) 0 0 0 0 1 (25) 1 (25) 2682 (102) 18.4 (3.3)
First Trimester (N=16) 39.1 (0.8) 1 (6) 1 (6) 3 (19) 2 (13) 3 (19) 5 (31) 3324 (328) 60.2 (22.1)
Second Trimester (N=11) 37.8 (3.2) 1 (9) 1 (9) 1 (9) 1 (9) 1 (9) 1 (9) 3412 (524) 59.0 (36.8)
Unknown Trimester (N=25) 38.2 (2.2) 1 (4) 1 (4) 1 (4) 2 (8) 3 (12) 3 (12) 3111 (676) 54.3 (29.5)
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Numbers reflect the mean (standard deviation) or N (%) with Z-score (as indicated) for the entire cohort and also by time of possible ZIKV exposure. Data for prenatal diagnosis of microcephaly and IUGR is based on the last ultrasound obtained per subject. AC, abdominal circumference; FL, femur length; HC, head circumference; IG-21, 2014 International Fetal and Newborn Growth Consortium for the 21st Century; WHO-FGC, World Health Organization Fetal Growth Chart study.

*

Birthweight data was available for 48 infants (preconception, N=2; first trimester, N=15; second trimester, N=8; unknown trimester, N=23).

Pregnancy outcomes were available in 52 of 56 cases (Table S6). In three pregnancies (3/52; 6%), a pregnancy termination was performed in the second trimester after a diagnosis of microcephaly. One stillbirth occurred at 30 weeks gestation (1/52; 2%) in a microcephalic fetus with symmetric severe growth restriction. Of the remaining 48 pregnancies, term birth occurred in 92% (44/48) and preterm birth in 8% (4/48). A postnatal head ultrasound was performed in 39 cases and identified a grade 1 intraventricular hemorrhage (1/39, 3%) or choroid plexus cyst (4/39, 10%), but no other structural findings associated with the congenital ZIKV syndrome (Table S6). Neonatal HC was measured in 47 of the 48 newborns with a mean Z-score of 0.4 using IG-21. At birth, microcephaly was observed in one neonate (HC Z-score ≤ −2) and no neonates had a HC Z-score ≤ −3 (Table S6). Interpretation of the laboratory testing for ZIKV infection of the neonate is limited by the transient nature of the viremia, but results were available for 41 infants; a possible ZIKV infection was diagnosed in 39% of cases (16/41, Table S4) and one infant had a confirmed ZIKV infection (1/41, 2%).

Microcephaly and Femur-Sparing Pattern of IUGR Identified using Single Fetal Biometric Measures and Fetal Body Ratios

Next, we compared paired biometric measures from each subject to determine if maternal ZIKV infection was associated with differential growth of the HC or AC with respect to the FL. Overall, the AC was significantly smaller than FL based on the last ultrasound scan in pregnancy by either IG-21 or WHO-FGC (Tables 2 and S7, p<0.001 for both analyses); this difference was also significant in every strata starting with the 24–27 6/7 week category for IG-21 and most strata for WHO-FGC. The HC was also significantly smaller than FL in the overall analysis by IG-21 (p<0.001) and in every strata beginning with 28–33 6/7 weeks; this difference was not significant by WHO-FGC.

Table 2.

IG-21 Fetal Z-Scores for Biometric Measures by Gestational Age Strata

Gestational Age Strata HC AC FL P values
FL vs. HC FL vs. AC
All (N=56) 0.1 (1.2) 0.0 (1.3) 0.7 (1.4) <0.001 <0.001
>34 weeks (N= 46) 0.4 (0.6) 0.2 (0.8) 0.9 (0.9) <0.001 <0.001
28 – 33 6/7 weeks (N= 38) 0.1 (1.4) 0.1 (1.4) 0.7 (1.4) 0.007 <0.001
24 – 27 6/7 weeks (N= 17) 0.5 (0.7) −0.1 (0.7) 0.6 (0.8) 0.8 0.002
18–23 6/7 weeks (N= 19) 0.2 (1.3) 0.0 (1.5) 0.5 (1.2) 0.9 0.7
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Values reflect Z-scores within each gestational age strata using the last US scan in each pregnancy or gestational age strata based on the number of subjects. HC, head circumference; AC, abdominal circumference; FL, femur length. P values were calculated using Wilcoxon rank sum to compare paired Z-scores (IG-21) between FL and HC or FL and AC. A p value of <0.05 was considered significant.

Another method to identify ZIKV-associated differential growth of the fetal head or abdomen with respect to the femur would involve an analysis of fetal body ratios (e.g. HC:FL or AC:FL). To this end, we developed IG-21 fetal body ratios based on previously published data from 4,607 normal pregnancies in 18 different countries.[21] These fetal body ratios were used to generate Z-scores in our cohort to compare differences in size of the fetal head and/or abdomen versus the femur. In contrast to a 5% rate of microcephaly, a femur-sparing pattern of fetal growth restriction was observed after 34 weeks gestation in 37% (17/46) of pregnancies based on either a small head (HC:FL; 28%, 13/46) or abdomen (AC:FL; 20%, 9/46) in relation to the femur (Z-scores <−1.3; Fig. 1). If we considered ultrasound data from any time during pregnancy, 52% (29/56) of pregnancies had a differentially small head or abdomen in comparison to the femur [Z-scores <−1.3; HC:FL 39% (22/56) and/or AC:FL 30% (17/56); Fig. 1]; this final analysis allowed inclusion of fetuses from the second trimester pregnancy terminations and the stillbirth and preterm birth cases. If we considered only women with symptomatic ZIKV infection, an abnormal HC:FL ratio was observed in 46% (6/13) and an abnormal AC:FL ratio in 15% (2/13). In pregnancies with an abnormal HC:FL or AC:FL ratio, the ratio became more skewed over time in most pregnancies (Fig. S1 and S2). Overall, the majority of pregnancies in our study with a possible maternal ZIKV infection developed a femur-sparing profile of growth restriction using fetal body ratios developed from the IG-21 sonographic standard.

Figure 1.

Figure 1

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Fetal Body Ratio Z-Scores from U.S. Women with Possible Maternal ZIKV Exposure Using the IG-21 Sonographic Standard. A negatively skewed distribution of HC:FL and AC:FL is apparent within every gestational age strata. Data is color coordinated to show individual subjects. Depending on the number of ultrasound scans per subject, one subject may contribute ultrasound data to multiple gestational age strata in the table, but only one (the latest) ultrasound per subject was used in each strata. Application of the IG-21 sonographic standard to generate Z-scores is shown for HC:FL (A), and AC:FL (B).

Comment

Principal Findings of the Study

Our study is the first to demonstrate a femur-sparing pattern of IUGR in late gestation of women with a possible ZIKV infection. This unusual fetal growth profile was found by application of the IG-21 and WHO-FGC standards and differs from prior models of IUGR (Fig. 2). We found a significant skewing of fetal biometrics with a smaller AC versus FL, which was first apparent in the 24–27 6/7 week strata. Fetal body ratios (HC:FL and AC:FL, by IG-21) were consistent with a femur-sparing pattern of fetal growth restriction in the majority of pregnancies with possible maternal ZIKV infection.

Figure 2.

Figure 2

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Femur-sparing Profile of IUGR in Comparison to Normal and Other Abnormal Fetal Growth Patterns. Aberrant fetal growth in association with a possible maternal ZIKV infection is characterized by a femur-sparing profile of aberrant fetal growth. This figure illustrates how the femur-sparing profile of IUGR compares to normal fetal growth and more common IUGR growth patterns (symmetric and asymmetric IUGR).

Results in the Context of What is Known

Fetuses that were either small for gestational age or growth restricted were reported to occur in 9% of pregnancies with a possible ZIKV infection in Rio de Janeiro, Brazil.[16] Interestingly, the authors characterized 4 cases of microcephaly in their cohort as either “proportionate” (2/4, 50%) or “disproportionate” (2/4, 50%) relative to the size of the infant; a “disproportionate” microcephaly indicated a grossly differential growth of the head with respect to other body parts in at least half of their index cases. IUGR has also been described as a hallmark feature of several murine models of ZIKV infection in pregnancy and is associated with spontaneous abortion and stillbirth in these models.[41, 42, 43, 44] Although a femur-sparing pattern of growth restriction has been mentioned in the literature[45], it has not been characterized in the context of maternal complications of pregnancy or exposure to any teratogenic virus. Interestingly, few studies have characterized the IUGR phenotype in pregnancies with viral infections with the exception of a symmetric profile of IUGR associated with congenital cytomegalovirus infection.[13]

Skewed Distribution of Fetal Biometry in Pregnancies with Possible Maternal ZIKV Infection

Beginning in the late second trimester, maternal ZIKV infection was associated with a significantly smaller AC, by both IG-21 and WHO-FGC, and HC by IG-21 compared to FL. Analysis of IG-21 fetal body ratios with respect to FL revealed a femur-sparing profile of growth restriction in the majority of pregnancies with a possible ZIKV infection. The stable or negative trajectory of the AC:FL or HC:FL over time and the high proportion of women with symptoms (nearly half) with an abnormal HC:FL ratio is concerning for ZIKV-associated fetal injury. Identification of a femur-sparing profile of fetal growth restriction using IG-21 fetal body ratios could aid pediatricians in prioritizing neonates for imaging in low-resource settings. It is important to note that this profile of injury may not be obvious using other sonographic standards, primarily due to differences in FL distribution. For example, the Hadlock sonographic standard is anecdotally associated with the finding of “short femurs” and may not yield the same growth restriction profile.[28, 29, 30] A discordance between the rate of fetuses with a small AC and rate of small for gestational age neonates may be a consequence of this particular type of growth restriction that preserves skeletal growth, which may compensate for birth weight. Whether abnormal growth of the fetus in relation to the femur correlates with long-term adverse outcomes for the developing child is unknown, but identification of an abnormal fetal body ratio (AC:FL or HC:FL) may be superior to measurement of fetal BPD or HC alone as a marker for ZIKV-associated fetal injury.

Clinical and Research Implications

The pathogenesis of perinatal infections resulting in fetal injury is complex and involves both indirect and direct effects. ZIKV infections could have a direct effect on fetal growth through targeted injury of the brain and liver, but also an indirect effect through trophoblast injury and a reduction in oxygen carrying capacity.[46] If viral tropism for cells in the fetal brain and liver is greater than tropism for the skeleton, this could produce differential viral effects on fetal growth that might result in the femur-sparing profile of fetal growth restriction that we observed in our study. As the size of the fetal abdomen directly correlates with liver size [47], ZIKV injury of the fetal liver may depress growth of the abdomen. ZIKV RNA has been detected in the liver in humans and animal models.[11, 48, 49] Liver injury is also a well-known outcome for many viruses related to ZIKV (e.g. Hepatitis C, dengue virus).[50, 51] Future studies of the effect of ZIKV on the fetal liver may in part explain the pathogenesis of fetal growth restriction with this infection.

We would like to emphasize that our results do not suggest that a femur-sparing profile of growth restriction is the only possible phenotype or outcome of perinatal ZIKV infection. A normal growth profile may occur if the pregnant woman clears the virus before vertical transmission can occur. A fetal growth profile consistent with symmetric IUGR may occur with early and severe placental infections, which could compromise placental function; this effect would be similar to observations of placental infarctions and compromised placental oxygen transport in a nonhuman primate model following experimental ZIKV infection.[46] Additional research may further elucidate the relationship between IUGR and ZIKV infection, and characterize extreme cases of fetal injury, phenotype of IUGR and impact of timing of infection. Finding a more sensitive biomarker of viral injury, such as a sonographic profile of fetal growth, may help guide the pediatricians’ evaluation and triage cases for postnatal follow up where resources are limited.

Strengths and Weaknesses

The strengths of this study are in the detailed fetal growth assessment from a relatively large sample of pregnancies with possible maternal ZIKV infection and the novel identification of a variant in fetal growth restriction associated with viral infection. A further strength is in the evaluation and comparison of biometric measures using two contemporary, international fetal growth studies. Finally, the novel use of IG-21 fetal body ratios to interpret fetal size in pregnancies with possible ZIKV infection may be useful for clinical care and also relevant to more common forms of IUGR. One limitation of our study is that the diagnosis of ZIKV infection is challenging due to the transient nature of viremia and cross-reactivity with other flaviviruses. Another important study limitation is the small sample size and lack of a specific fetal growth standard for this population; creating a robust standard would be challenging, however, given the ethnic diversity of the cohort. Future studies with larger cohorts are necessary to validate our findings and determine if adverse neonatal outcomes might be associated with a femur-sparing profile of growth restriction. Although our study definitions of IUGR and microcephaly were in line with current standards, they may capture some constitutionally small infants; as we did not base IUGR on EFW, this may also limit comparability to other studies. However, the surprising distribution of cases with differential growth of the abdomen and head versus the femur is suggestive of an unusual pattern of fetal growth restriction that is not typically seen in pregnancy.

Conclusion

In summary, our results suggest that infants born following a possible maternal ZIKV infection may have abnormal growth patterns of the fetal head and abdomen with respect to the femur. Calculation of IG-21 fetal body ratios (AC:FL or HC:FL) may provide an early indication of aberrant fetal growth before a clinical or sonographic diagnosis of IUGR or microcephaly. Alerting clinicians to deviations in symmetric growth of a nonmicrocephalic fetus with congenital ZIKV exposure may aid in the identification of cases at risk for a greater spectrum of ZIKV-associated morbidity (e.g. eye abnormalities, postnatal microcephaly). These cases could be prioritized for more intensive neonatal follow-up in low resource settings for earlier interventions after delivery. Ultimately, larger cohorts will be important to validate a femur-sparing profile of growth restriction in women with a possible ZIKV infection in pregnancy and investigate whether this profile might predict adverse fetal and neonatal outcomes.

Supplementary Material

Implications and Contributions.

A. Why was this study conducted?

To determine if Zika virus infection during pregnancy is associated with a femur-sparing pattern of fetal growth restriction, similar to observations in a nonhuman primate model of decelerating growth of the fetal head and abdomen with respect to femur length.

B. What are the key findings?

An unusual femur-sparing pattern of fetal growth restriction was detected in the majority of fetuses with congenital ZIKV exposure using Intergrowth-21st Project fetal body ratios comparing head or abdominal circumference to femur length.

C. What does this study add to what is already known?

Fetal body ratios may provide a new screening tool to detect Zika virus-associated fetal injury in pregnancies without overt microcephaly.

Acknowledgments

This work was primarily supported by generous private philanthropic gifts mainly from five donors in Florida, who wish to remain anonymous. Further support was obtained from the University of Washington Department of Obstetrics & Gynecology, Seattle Children’s Research Institute and the National Institutes of Health, Grant # R01AI100989 (L.R and K. A. W), AI083019 (M.G. Jr.) and AI104002 (M.G. Jr.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other funders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We would like to acknowledge Jan Hamanishi for technical assistance with preparation of the figures. We thank Dr. Torvid Kiserud for consultation and advice related to the WHO Fetal Growth Charts.

Glossary of Terms

AC

abdominal circumference

BPD

biparietal diameter

CDC

Centers of Disease Control

FL

femoral length

HC

head circumference

IG-21

2014 International Fetal and Newborn Growth Consortium for the 21st Century

IUGR

intrauterine fetal growth restriction

NICHD

Eunice Kennedy Shriver National Institute of Child Health and Development

PRNT

plaque reduction neutralization test

RT-PCR

real-time polymerase chain reaction testing

WHO

World Health Organization

WHO-FGC

World Health Organization Fetal Growth Chart

ZIKV

Zika virus

Footnotes

The authors report no conflict of interest.

Data Availability

Fetal biometric measures from de-identified cases will be made available upon request.

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Contributor Information

Christie L. WALKER, Seattle, WA; Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine, University of Washington.

Audrey A. MERRIAM, New York City, NY; Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Columbia University Medical Center.

Eric O. OHUMA, Oxford, United Kingdom; Nuffield Department of Medicine, Centre for Tropical Medicine and Global Health, University of Oxford; Centre for Statistics in Medicine, Nuffield Department of Orthopaedics, Rheumatology & Musculoskeletal Sciences, University of Oxford.

Manjiri K. DIGHE, Seattle, WA; Department of Radiology, University of Washington.

Michael GALE, Jr., Seattle, WA; Center for Innate Immunity and Immune Disease, Department of Immunology and Department of Global Health, University of Washington.

Lakshmi RAJAGOPAL, Seattle, WA; Center for Innate Immunity and Immune Disease, Department of Pediatrics, University of Washington; Center for Global Infectious Disease Research, Seattle Children’s Research Institute.

Aris T. PAPAGEORGHIOU, Oxford, United Kingdom; Nuffield Department of Obstetrics & Gynaecology and Oxford Maternal & Perinatal Health Institute, Green Templeton College, University of Oxford.

Cynthia GYAMFI-BANNERMAN, New York City, NY; Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Columbia University Medical Center.

Kristina M. ADAMS WALDORF, Seattle, WA; Department of Obstetrics & Gynecology, Center for Innate Immunity and Immune Disease, and Department of Global Health University of Washington; Sahlgrenska Academy, Gothenburg University, Sweden.

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