Reproductive Toxicology 85 (2019) 6–11

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Reproductive Toxicology
journal homepage: www.elsevier.com/locate/reprotox

Screening for prenatal alcohol exposure and corresponding short-term T

neonatal outcomes
⁎
Stefan Maxwella, , Stephanie Thompsonb, Fadi Zakkoc, Luis A. Bracerod,1
a
Pediatrix Medical Group and Department of Pediatrics, Charleston Area Medical Center, Charleston, WV, USA
b
Center for Health Services and Outcomes Research, Charleston Area Medical Center Health Education and Research Institute, Charleston, WV, USA
c
Department of Pediatrics, Charleston Area Medical Center, Charleston, WV, USA
d
Department of Obstetrics and Gynecology, West Virginia University-Charleston Division, Charleston Area Medical Center, Charleston, WV, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Detection of prenatal alcohol exposure (PAE) is important for early intervention and treatment. The main
Alcohol biomarker purpose of this study was to compare 1.) PAE rates using the biomarker, phosphatidylethanol (PEth), in um-
Neonatal outcomes bilical cord (UC) blood vs. ethyl glucuronide (EtG) in UC tissue, the standard of care, and 2.) Pregnancy char-
Fetal alcohol spectrum disorder acteristics and neonatal outcomes in newborns positive vs. negative for PAE biomarkers. We examined records of
Neurodevelopmental disabilities
neonates born over a two-year span receiving UC-PEth dried blood spots testing at the time of delivery in
addition to standard of care PAE screening (n = 146). UC-PEth testing had a higher PAE detection rate (26%) vs.
UC tissue EtG (0%, p < 0.01). PAE was not associated with any neonatal dysmorphic features or short-term
adverse outcomes. The absence of significant clinical findings for identifying PAE in neonates reinforces alcohol
biomarker necessity. We conclude that UC-PEth may be a valuable test for assessing PAE at birth and in iden-
tifying infants at risk for developing fetal alcohol spectrum disorder.

1. Introduction the risk of these secondary disabilities.

Prenatal alcohol exposure (PAE) remains a public health challenge
and can result in a variety of fetal defects; with the magnitude, timing,
and duration of alcohol exposure influencing outcome severity [1–3].
One of the most severe PAE consequences is the diagnosis of fetal al-
cohol syndrome (FAS), a disorder defined by growth deficiencies, spe-
cific facial features, and nervous system malformations. Fetal alcohol
spectrum disorders (FASD) is an umbrella term encompassing a range of
adverse effects associated with PAE, including not only FAS, but also
the diagnoses of partial fetal alcohol syndrome (pFAS), alcohol-related
birth defects (ARBD), alcohol-related neurodevelopmental disorder
(ARND), and neurobehavioral disorder associated with prenatal alcohol
exposure (ND-PAE) [4]. FASD prevalence is estimated to be as high as
1–5% of schoolchildren [5], making PAE the most preventable source of
birth defects and intellectual disability in the United States [4]. The
consequences of PAE can be lifelong. Compared to the general popu-
lation, individuals with FASD are at increased risk for psychiatric,
emotional, and behavioral problems, as well as substance abuse, school
suspensions and expulsions, not completing high school, and criminal
misconduct [6]. Delayed diagnosis or misdiagnosis of FASD increases
Identification of infants exposed to alcohol in utero is crucial to
making early FASD diagnoses. Timely diagnosis is the key to providing
early interventions and treatments, particularly in order to take ad-
vantage of the neuroplasticity occurring prior to three years of age. Two
basic methods are used to identify newborns exposed to alcohol in
utero: self-reporting in birth mothers or biomarkers in specimens from
the birth mother or neonate. Unfortunately, self-reporting suffers from
problems with informant’s veracity and recall accuracy. A meta-ana-
lysis of eight studies found PAE prevalence rates to be ˜4 times higher
with meconium biomarker testing compared to corresponding maternal
self-reports [7]. Thus, objective biological alcohol markers are crucial
to diagnose PAE in the early postnatal period.
Several biochemical compounds have shown usefulness in PAE de-
tection. Phosphatidylethanol (PEth) is one such biomarker, an ab-
normal cellular membrane phospholipid formed only in the presence of
ethanol [8]; and is highly sensitive and specific to alcohol exposure and
has a detection window of more than 2 weeks [9,10]. PEth is considered
to be insensitive to incidental ethanol exposures, such as mouthwash
and antibacterial hand sanitizers [9,11,12]. This biomarker has a higher
sensitivity for alcohol exposure compared to the traditional alcohol

⁎
Corresponding author at: Suite 406, 830 Pennsylvania Avenue Charleston, WV, 25302, USA.
E-mail address: neodoc1124@gmail.com (S. Maxwell).
1
Present address: Department of Obstetrics and Gynecology, Hofstra Northwell School of Medicine, Southside Hospital, Bay Shore, NY, USA.

https://doi.org/10.1016/j.reprotox.2019.01.009
Received 10 August 2018; Received in revised form 19 December 2018; Accepted 17 January 2019
Available online 18 January 2019
0890-6238/ © 2019 Published by Elsevier Inc.
S. Maxwell et al.
markers of carbohydrate-deficient transferrin, γ-glutamyl transferase,
and mean corpuscular volume [9] and the direct ethanol metabolite,
fatty acid ethyl esters [13]. PEth has been previously detected in um-
bilical cord tissue [14] and residual dried blood spots (DBS) collected
for routine newborn screening [15,16], but has not been reported using
umbilical cord DBS. Umbilical cord blood is easy and noninvasive to
collect, with larger volumes of blood from the umbilical cord available
compared to heel stick.
The main purpose of our study was to determine if umbilical cord
blood PEth (UC-PEth) testing identifies an increased number of neo-
nates exposed to alcohol in utero compared to our institution’s current
PAE screening method, ethyl glucuronide (EtG), in umbilical cord
tissue. Additionally, we wished to determine if PAE detected at the time
of birth was associated with short-term adverse neonatal outcomes.
Multiple studies demonstrate increased risk for low birthweight and
premature delivery following PAE, especially in neonates born to
women who consumed alcohol heavily or partook in binge drinking
during pregnancy [3,17,18]. In addition to birth weight and gestation
duration, we examined short-term outcomes commonly associated with
premature birth such as infection, respiratory distress, intraventricular
hemorrhage, hyperbilirubinemia, and electrolyte abnormalities [19].
2. Material and methods
2.1. Design
We examined the records of neonates born at a tertiary care medical
center (Charleston Area Medical Center-Women and Children’s
Hospital, Charleston, WV) over a two-year time span that received UC-
PEth DBS testing. The study sample was comprised of consecutive
neonates born between the dates of 1/1/2013 and 2/28/2015 to
women receiving obstetrical care at an on-site clinic whose care in-
corporated a quality improvement initiative examining novel strategies
for PAE detection. The initiative of using PEth DBS testing was in ad-
dition to the standard of care prenatal alcohol and substance use
screens utilized by the obstetrical clinic and hospital. This initiative and
PEth testing of women during early pregnancy at the first prenatal visit
are described in Bracero et al [20].
UC-PEth testing results were not used for clinical care. Each new-
born was assigned a research ID as no identifiers were kept with the
specimen. UC-PEth test results were not available to obstetrical or pe-
diatric care providers. Newborns without prenatal care and stillborn
infants were excluded from analysis. All aspects of the study were re-
viewed and approved by the CAMC/West Virginia University,
Charleston Division, Institutional Review Board. A waiver of consent
was granted by the Review Board as the study was a quality improve-
ment initiative and the standard of care includes substance use
screening.
2.1.1. Umbilical cord blood collection
Umbilical cord blood was obtained immediately after birth by
nursing staff and was collected on blood spot screening cards provided
by United States Drug Testing Laboratories (USDTL, Des Plaines, IL,
USA). Umbilical cord blood specimens were collected within the first
5 min following birth to avoid clotting. One drop of blood from an
umbilical cord segment was placed into each of five circles on the
provided filter paper card. The specimen cards were then placed in the
drying box provided by USDTL and closed using a tamper-proof seal.
Specimens were dried for a minimum of one hour at room temperature
before storage at −20 °C until shipment to USDTL.
2.2. Umbilical cord blood PEth analysis
DBS specimens were analyzed for PEth levels at USDTL using pre-
viously published methods [16,20,21]. The extraction and detection of
PEth from the umbilical cord DBS were completed using the liquid
7
Reproductive Toxicology 85 (2019) 6–11
chromatography-tandem mass spectrometry (LC–MS/MS) method. In
brief, three standard blood spots (3.1 mm) were prepared for each
specimen, as well as a calibrator and control. The punches were ex-
tracted with methanol (1 ml), evaporated under nitrogen, and then
reconstituted into 1.0 ml of mobile phase A (50% 2 mM ammonium
acetate: 25% acetonitrile: 25% isopropanol). Separation was achieved
via high-pressure liquid chromography (Eksigent HALO C-8 column)
and detection performed using AB Sciex 5500 tandem mass spectro-
meter. A single isomer of PEth (palmitoyl/oleoyl), the most prevalent
PEth species, was measured. The limit of detection was 2.00 ng/mL, the
limit of quantitation was 8.00 ng/mL, and the assay was linear up to
800.00 ng/mL. PeTH concentrations were recorded, with positive PEth
screens being defined as blood spots having values greater than the
previously reported PEth cutoff concentration of 8.00 ng/mL [16,20].
2.3. Umbilical cord tissue EtG assay
Per hospital policy, umbilical cord tissue was tested for alcohol and
other substances if there was a positive maternal history for drug ex-
posure or at physicians’ discretion. The umbilical cord EtG assay
(USDTL) was obtained at the time of delivery. Cord EtG assay screens
for the direct alcohol biomarker, EtG, in the UC forming from the
conjugation of ethanol with activated glucuronic acid [22]. For this
assay, an umbilical segment approximately 10 cm long was obtained at
the time of birth. Cords were stripped of blood, lightly rinsed in sterile
saline, placed in sterile plastic specimen containers, and then frozen for
subsequent shipment to USDTL and processed according to their stan-
dard procedures [12]. The limit of detection was 1 ng/g and the limit of
quantitation was 3 ng/g for the cord EtG assay.
2.4. Data collection
To examine associations between PAE and neonatal characteristics
and short-term outcomes, medical records of newborns and corre-
sponding birth mothers were retrospectively reviewed. We obtained
basic demographics on birth mothers, including age, race, marital and
education status, insurance type (private, public or self-pay) and history
of prior births. We acquired maternal alcohol use information from the
initial prenatal clinic visit. Birth mothers were screened using the
standard American College of Obstetricians and Gynecologists prenatal
record questionnaire inquiring about their alcohol intake during preg-
nancy and a urine ethanol test (Ameritox, Baltimore, MD, USA). Data
on neonates’ weight, height, head circumference, and estimated gesta-
tional age at birth was collected. Preterm birth was defined as a birth
occurring prior to 37 weeks of gestation. The associated percentile for
birthweight, height, and head circumference for gestational age were
determined using 2003 Fenton Growth Charts [23]. Small for gesta-
tional age was defined as below the 10th percentile for gestational age.
Apgar scores were obtained at 1 and 5 min post-delivery and stratified
as being ≥ 7 or < 7. If the newborn infant received care in the neonatal
intensive care unit (NICU), this was recorded. All newborns were ex-
amined by pediatricians for growth restriction and dysmorphic features
associated with the FASD (smooth philtrum, thin upper vermilion
border, small palpebral fissures, etc.) [4]. We obtained short-term ad-
verse outcomes for neonates including a diagnosis of sepsis, necrotizing
enterocolitis (any stage), respiratory distress syndrome, hypoglycemia
(< 43 mg/dl) /hyperglycemia > 135 mg/dl), hypocalcemia
(< 7.8 mg/dl), hyperbilirubinemia (> 3.9 mg/dl), or intraventricular
hemorrhage (any grade).
2.5. Statistical analysis
SPSS (Version 19.0) was used for all data analysis. Descriptive sta-
tistics included frequencies and percentages for categorical variables,
while means +/- standard deviations (SD) were used for continuous
variables unless otherwise noted. Continuous variables were compared
S. Maxwell et al. Reproductive Toxicology 85 (2019) 6–11

Fig. 1. Distribution of PEth concentrations in study patients. The limit of quantitation was 8.00 ng/mL.

between neonates having positive UC-PEth blood spot testing to neo- Table 1
nates with negative PEth test results by using student independent Maternal Characteristics.
samples t-tests or Mann-Whitney U, if data was not normally dis- Positive PEth Negative PEth P value
tributed. Categorical variables were reported as percentages and were testing testing
compared using Chi-square or Fisher’s Exact Test when more than 20% N = 43 N = 119
of cells had expected counts less than five. McNemar’s test for paired
Maternal Age 23.0 ± 5.3 24.3 ± 5.4 0.19
proportions was employed to detect a difference between the two al- Race 0.90+
cohol screens (UC-PEth and cord EtG) for PAE detection. A p-level White 36 (83.7%) 94 (79.0%)
of < 0.05 was considered statistically significant. Black 7 (16.3%) 23 (19.3%)
Other 0 (0%) 2 (1.7%)
Married 4 (9.3%) 21 (17.6%) 0.19
3. Results Education level > 12 years 16 (37.2%) 34 (28.6%) 0.29
Insurance Status 1.00+
A total of 292 neonates were born during the study period to 289 Public 39 (90.7%) 107 (89.9%)
Private 2 (4.7%) 7 (5.9%)
women who received prenatal care at the study obstetrical clinic and Self-pay 2 (4.7%) 5 (4.2%)
were included in the quality improvement initiative to assess PAE de- Nulliparous 16 (37.2%) 43 (36.1%) 1.00
tection. Of those, 168 neonates had UC-PEth dried blood spot testing at Screening of maternal alcohol consumption at initial obstetrical visit
the time of delivery, with 6 of those neonates being excluded due to Self-reporting alcohol use during pregnancy (n = 162)
Self-reported alcohol use N = 43 N = 119 0.29+
improper collection. Of the 162 neonates included in the analysis, 43
during pregnancy 2 (4.7%) 2 (1.7%)
(26.5%) had positive UC-PEth bloodspots (≥ 8 ng/ml). Fig. 1 shows a Birth mothers with urine toxicity screen (n = 156)
histogram of the distribution of PEth results. The mean PEth level was Positive for alcohol N = 42 N = 114 0.56+
63.34 ng/ml with a range of 8.14–1260.00 ng/ml and a median value of 0 (0%) 3 (2.6%)
16.55 ng/ml. Birth mothers with PEth testing at initial prenatal visit
(n = 108)
As per standard of care during the study period, 146 of the 162 Prenatal positive PEth testing N = 26 N = 78 0.68+
umbilical cords were concurrently tested for PAE using the cord tissue 1 (3.8%) 7 (9.0%)
EtG assay. All cord tissue samples were negative for EtG. Thus, in the
+
neonates receiving the two alcohol screens, UC-PEth testing had a sig- Fisher’s exact test.
nificant higher detection rate for PAE, with 38 of 146 (26%) newborns
having a positive PEth screen versus 0 out of 146 (0%) newborns with 10th percentile for gestational age) birthweight, length, and head cir-
the cord tissue EtG assay (McNemar's test, p < 0.001.) cumference, very few neonates met this definition (< 3%), with the
The birth mother characteristics of age, race, marital status, edu- distribution being similar between PAE cohorts (p = 1.00). Apgar
cation level, insurance status, and history of prior births did not sig- scores obtained at 1 and 5 min post birth, as well as the need for NICU
nificantly differ between neonates having positive UC-PEth DBS and care, did not differ between neonates with positive and negative PAE
those with negative blood spots (Table 1). Additionally, detection rates screens.
of maternal alcohol consumption in early pregnancy (mean estimated We wished to determine if a positive PAE screen at birth was as-
gestational age at initial prenatal visit: 11.3 ± 9.1 weeks) in our study sociated with short-term adverse neonatal outcomes. Rates of sepsis,
cohort was 2.8% when using self-reporting, 1.9% with urine screening, necrotizing enterocolitis, respiratory distress syndrome, in-
and 7.7% with positive maternal PEth DBS (≥ 8 ng/ml). PAE, as traventricular hemorrhage, hypoglycemia/ hyperglycemia, and hy-
measured by UC-PEth testing, did not associate with maternal alcohol perbilirubinemiain the neonates did not differ between neonates with
consumption in early pregnancy (i.e. the initial obstetrical visit) as positive and negative PAE screens (Table 3). No newborn infants ex-
detected by self-reporting (p = 0.29), urine toxicity screens (p = 0.56), perienced hypocalcemia. Additionally, no neonates had FAS-associated
or peripheral blood PEth testing (p = 0.40). facial features documented in their medical record.
Delivery method and as well as the birth outcomes of estimated
gestational age at delivery and birth weight did not differ between 4. Discussion
neonates with positive and negative UC-PEth results (Table 2). Rates of
premature birth (< 37 weeks) did not differ between newborns having As PAE can result in a variety of lifelong neurodevelopmental,
a positive PAE screen (6/43, 14.0%) versus those with negative screens cognitive, and behavioral disabilities, we wished to improve PAE
(16/119, 13.4%, p = 0.79). When examining small for age (below the identification strategies in neonates. Improving PAE detection should

8
S. Maxwell et al. Reproductive Toxicology 85 (2019) 6–11

Table 2 potential method for PAE detection due to ease of collection, transport,
Pregnancy and Neonatal outcomes stratified by prenatal alcohol exposure. storage and processing of samples, lower analysis cost, and high sen-
Positive PEth Negative PEth P value sitivity for ethanol exposure [29,30]. Women with blood PEth con-
testing testing centrations greater than 4 nM and who self-reported alcohol con-
N = 43 N = 119 sumption in the first trimester had increased risk for spontaneous
abortions [31]. Additionally, previous studies have analyzed PEth levels
Delivery Method
C-section 7 (16.3%) 32 (26.9%) 0.16
in DBS obtained as part of routine, state-mandated, newborn screening
Vaginal 36 (83.7%) 87 (73.1%) as a means to measure PAE. In a statewide prevalence study of Texas,
Gestational Age PAE (defined as PEth levels > 20 ng/ml) was found in 8.4% of the
≤30 weeks 1 (2.3%) 2 (1.7%) 0.09 1000-card sample obtained previously for newborn genetic screening
31-33 weeks 3 (7.0%) 4 (3.4%)
[15]. Another study looking at prevalence found that in a sample of 201
34-36 weeks 2 (4.7%) 10 (8.3%)
37-39 weeks 34 (79.1%) 76 (63.9%) infants born at the University of New Mexico Hospital (Albuquerque,
≥40 weeks 3 (7.0%) 27 (22.7%) NM), 6.5% tested positive (> 20 ng/ml) for PEth [28], while a study of
Birthweight 250 cards obtained from a Midwestern state shown much lower pre-
extreme (< 1000 g) 0 (0%) 1 (0.8%) 0.66
valence (4% with PEth ≥ 8 ng/ml and 1.6% with PEth > 20 ng/ml)
very low (1000-1499 g) 0 (0%) 2 (1.7%)
low (1500-2500 g) 7 (16.3%) 13 (10.8%)
[14]. Compared to above-mentioned studies, we found higher rates of
normal (> 2500 g) 36 (83.7%) 103 (86.6%) PAE, with 26% having positive PEth blood spots (≥ 8 ng/ml) and 12%
Low birthweight (< 10%) for 1 (2.3%) 3 (2.5%) 1.00+ when using the higher cut-off of > 20 ng/ml.
gestionational age While measuring PAE via PEth levels in heel stick DBS has been
Low height (< 10%) for 0 (0%) 2 (1.7%) 1.00+
shown to be a reliable and convenient method for specimen collection,
gestionational age
Low head circumference (< 10%) 1 (2.3%) 3(2.5%) 1.00+ using umbilical cord blood for PEth measurement is an additional, al-
for gestionational age ternative specimen to use in PAE detection. Umbilical cord blood has
Low (< 7) Apgar Score at 1 minute 1 (2.3%) 6 (5.0%) 0.68+ the benefit of being available immediately after birth, allowing for
Low (< 7) Apgar Score at 5 minutes 1 (2.3%) 1 (0.8%) 0.46+ 24–48 h earlier collection and returned results compared to heel stick
Required NICU stay 6 (14.0%) 13 (10.9%) 0.60
in patients with NICU stays NICU LOS 18.5 [10.5- 15.0 [5.8-39.0] 0.51*
DBS collected in conjunction with routine newborn screens. Earlier
(days), median [IQR] 32.0] testing may decrease the occurrence of false negatives resulting from
PEth degradation occurring during the 24–48 h post birth. Comparisons
+
Fisher’s exact test. between PEth levels in umbilical cord blood obtained at birth and PEth
* Mann-Whitney U. levels in specimens obtained from heel sticks collected 24–48 h post
birth will determine if earlier collection results in increased PEth con-
assist in the direction of at-risk infants and children to early childhood centrations and sensitivity for identifying PAE.
monitoring and interventions. A total of 26.5% of newborns included in Our study and other studies using PEth screening of newborn DBS
the study were found to be positive for PEth in cord DBS obtained at [15,16,29,30] indicate the need for further investigation regarding PAE
delivery, suggesting that these neonates had some level of PAE within prevalence. It appears that there may be an alarming number of neo-
the 3 or more weeks prior to birth. UC-PEth appears more sensitive for nates that have been exposed to alcohol during the weeks preceding
PAE detection than cord tissue EtG testing, with matched-pair analysis birth, which may have lasting consequences. None of the neonates in
of showing that more than a quarter of the neonates were positive for our study exhibited the morphological or neurological features of FAS.
PEth while no cord tissue specimens were positive for EtG. In addition Many of the symptoms and criteria for FASD diagnoses are not apparent
to possible differences in the two alcohol metabolites tests’ sensitivity in newborns. Studies confirmed that missed diagnoses and mis-
and specificity, differences exist in their detection windows, which may diagnoses of FASD are common, with Little et al showing that the FAS
partially explain the contrasting results of the two PAE screens. Serum diagnosis was missed and undocumented in 100% of newborns which
EtG in adults has been shown to have a relatively short half-life of 2–3 h were later identified as having the syndrome [32]. Additionally, FASD
and detection window of up to 80 h following alcohol consumption diagnoses were previously missed in 80% of foster and adopted youth
[22]. PEth’s half-life is approximately 4 days and is detectable in blood later diagnosed following mental health care referral [33] and Mays
for up to 2–4 weeks following the last drinking episode in non-pregnant et al found only 2 of 222 children with FASD were previously diagnosed
adults [24–26]. In pregnant women, PEth has been detected up to 6 with a PAE-related disorder prior to active case ascertainment as first-
weeks after the last self-reported alcohol consumption [27]. Also of grade students [3]. Thus, identifying infants with PAE can be difficult
note, EtG can be formed in vitro from endogenous ethanol present in and FASD diagnoses not occurring until childhood or early adolescence,
the sample or during sample collection [14] and at least in urine, EtG if at all. The challenge of diagnosing FASD in infants highlights the
levels can be altered post-collection by bacterial pathogens [28]. critical need for biomarker development and utilization for PAE de-
Due to these limitations associated with EtG and other alcohol tection and identification of at-risk infants.
markers, PEth DBS testing has generated significant interest as a

Table 3
Adverse neonatal outcomes stratified by prenatal alcohol exposure.
Positive PEth testing Negative PEth testing P value
N = 43 N = 119

Sepsis 4(9.3%) 7(5.9%) 0.48+

Necrotizing Enterocolitis 0 (0%) 1(0.8%) 1.00+
Respiratory Distress Syndrome 2 (4.7%) 5(4.2%) 1.00+
Intraventricular hemorrhage 0 (0%) 1(0.8%) 1.00+
Hyperbilirubinemia 8(18.6%) 11(9.2%) 0.10
Hypoglycemia or Hyperglycemia 3(7.0%) 12 (10.1%) 0.76+
Hypocalcemia No neonates had hypocalcemia
Fetal Alcohol Syndrome Facial Features No newborn infant had any documented Fetal Alcohol Syndrome facial features (smooth philtrum, thin upper vermilion, small palpebral fissures)

+
Fisher’s exact test.

9
S. Maxwell et al.
The rate of alcohol use occurring in late pregnancy was 26% as
detected via UC-PEth and was greater than rates of early pregnancy
maternal alcohol consumption as measured by self-reporting, urine
screens, or maternal PEth levels. The timing of alcohol consumption
during pregnancy may have implications for counseling strategies used
for PAE prevention as well as the resulting effects on neonatal devel-
opment. Maternal patterns of alcohol consumption and the timing of
PAE relative to critical development periods influence the extent of
central nervous system damage and subsequent cognitive and beha-
vioral deficits. It is generally accepted that heavy drinking is associated
with FAS and the adverse outcomes of growth restriction, birth defects,
and neurodevelopmental problems [4,34]. PAE during early gestation is
likely to result in structural neuro- and facial anatomical defects, while
later PAE is associated with neurocognitive deficits without facial
dysmorphia [3]. However, assessing the effects of PAE timing on the
subsequent effects in the neonate and child is difficult due to women
consuming alcohol in late pregnancy, as shown by UC-PEth, that may
have also consumed alcohol in the second and early third trimesters. In
an investigation of self-reported alcohol consumption patterns of birth
mothers of children diagnosed with FASD in comparison to non-FASD
controls, only 2 of 115 alcohol consuming women drank alcohol ex-
clusively in the third trimester, while 70 of 115 women consumed al-
cohol in all 3 trimesters, increasing FASD diagnosis likelihood 65 times
compared to abstainers [2].
PAE, as identified by UC-PEth testing, was not associated with any
maternal characteristics or neonatal outcomes. We were limited in
sample size due to constraints of the inclusion criteria and participation
in the quality improvement initiative; therefore the study was not
powered to identify differences in neonatal adverse events. Similar to
previous studies reporting on third trimester PAE [35,36], our study did
not find an association between PAE, as measured by UC-PEth levels,
and early neonatal adverse outcomes including low birthweight and
premature birth. We did not find an association between neonatal short-
term outcomes previously linked to PAE such as infection and sepsis
[37] and decreased Apgar scores [38]. Additionally, the adverse out-
comes of necrotizing enterocolitis, respiratory distress syndrome, glu-
cose dysregulation, hyperbilirubinemia and intraventricular hemor-
rhage did not associate with PAE. Further studies are needed to answer
questions related to patterns and levels of maternal alcohol consump-
tion during pregnancy and resulting PEth levels in newborn infants.
However, any detectable level of an alcohol metabolite in a newborn is
concerning, as no amount of PAE is viewed as safe [4]. PAE, even if not
generating evident morphological or neurobehavioral alterations in the
neonate, may still have harmful consequences that might not be iden-
tified or intervened upon for several years after birth.
One drawback of assessing PAE at birth is that damage to the ner-
vous system may have already occurred. However, the identification of
infants exposed to alcohol in utero is crucial to making an early FASD
diagnosis which would facilitate close monitoring of their development.
Early diagnosis is the key to providing timely interventions and treat-
ment, with early diagnosis being one of the strongest predictors for
improved outcomes in individuals with a FASD [6]. Interventions tar-
geting the cognitive, behavioral and learning impairments of FASD
have shown promising outcomes including prevention of poor perfor-
mance in school, mental health problems, substance abuse, de-
linquency, and incarceration [6]. The American Academy of Pediatrics
(AAP) endorses early identification of and intervention for FASD [4,39].
Increased monitoring of children at-risk for FASD could ease and fa-
cilitate entry in existing early intervention programs such Birth-to-
Three [40], Right from the Start [41], and established Attachment and
Biobehavioral Catch-Up [42,43] programs. Bertrand presented sum-
maries of five interventions demonstrating effectiveness for improving
outcomes in children (some as young as 3 years old) diagnosed with a
FASD [44]. However, recruitment of infants and toddlers as participants
in interventions can be challenging as children with FASD are often not
identified until school-age or later. Thus, improving identification
10
Reproductive Toxicology 85 (2019) 6–11
strategies for at-risk infants, toddlers, and preschool age children
should consequently help expand FASD early intervention development
and evaluation.
While PEth is a sensitive and specific biomarker of PAE, it has the
limitation of having a detection window of an estimated two to four
weeks from the last exposure. Meconium can serve as a valuable spe-
cimen for the PAE biomarkers of fatty acid ethyl esters (FAEEs), ethyl
glucuronide, and ethyl sulfate. Meconium is thought to have a detection
window from the second and third trimester due to its formation and
remaining stable throughout this time period [45]. However, com-
paring meconium- and cord-derived biomarkers for PAE was outside
the scope of our study. The sensitivity of newborn PEth-DBS appears
similar to meconium FAEEs, while meconium FAEEs have lower spe-
cificity [46]. Additionally, meconium collection can be difficult due to
the high proportion of specimens that are unusable for testing due to
passage in utero, delayed passage once born, or inadequate volume
[47].
The detection window and elimination rate of PEth in newborns is
unknown and has not been defined; the half-life of four days for PEth
and the window of detection for PAE are largely based on data from
studies on adult alcoholics. Given that metabolism of PEth in newborns
is largely unknown, the elimination window of PEth in newborns may
be either shorter due to the shorter lifespan of fetal and neonatal ery-
throcytes or longer if fetus/newborns have a lower capacity to meta-
bolize PEth than alcohol-consuming adults [29,30]. Thus, additional
studies are needed to better delineate neonatal PEth kinetics and
window of detection, including studies comparing PEth levels in DBS
obtained at birth with those obtained as part of routine newborn
screening (˜at 48 h post birth), and is the subject of future research at
our institution. Additionally, further investigations are needed to ex-
amine maternal alcohol consumption patterns using self-reporting and/
or repeated testing of alcohol biomarkers during gestation comparing
with neonatal PEth levels and outcomes. Lastly, we surmise that posi-
tive PEth tests at birth may predict neurological deficits as the children
mature, but have no data at this time to make this association between
PEth levels and long-term cognitive outcomes.
5. Conclusions
UC-PEth testing had a higher detection rate for PAE compared to
cord EtG screens. However, positive PEth test results did not associate
with maternal, neonatal, or outcome characteristics. The lack of phy-
sical characteristics and short-term adverse neonatal outcomes in
identifying neonates exposed to alcohol in utero supports the im-
portance of biomarkers. The idea that we are able to use a biomarker to
detect alcohol exposure for up to one month after consumption and to
identify infants at risk for PAE –related neurological deficits and be-
havioral aberrations is an extremely important concept. Placing these
at-risk infants in early targeted intervention programs may improve
outcomes.
Conflict of Interest
The authors report no conflict of interest.
Acknowledgments
The authors would like to thank Community Voices and West
Virginia Perinatal Partnership for funding this study, and United States
Drug Testing Laboratories for providing PEth testing materials. In ad-
dition, authors would like to thank Angel Cinco, Jennifer DePond,
Angela Henderson, April White, and Dara Seybold in assisting with
study coordination.
S. Maxwell et al.
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