1. Alcohol use in Pregnancy ? Under the Radar
Stefan R Maxwell MD FAAP
Director NICU CAMC Women & Children’s Hospital
PEDIATRIX MEDICAL GROUP

3. Disclosures I am not discussing any commercial products or services
I have no financial interests herein
I am in compliance with HIPAA in this presentation

4. Objectives
Fetal alcohol syndrome pFAS, ARND, ARBDs, ND-PAE, FASD Current methods of identification Misdiagnosis and Missed Diagnosis New research, Prevalence in WV Future directions??

5. Discovery of Fetal Alcohol Syndrome
1968 Paul Lemoine in Nantes, France recognized alcohol effects on the fetus, but was ignored by peers (127 children in 69 families)
1973 David Smith and Kenneth Jones working at Univ. of Washington in the area of Dysmorphology became curious…..
March of Dimes increased awareness of birth defects caused by teratogens
Working together they realized alcoholic mothers had children with developmental delays, microcephaly and growth deficiency
Described the Fetal Alcohol Syndrome

6. Historical evidence of effects of alcohol on developing fetus
Olegaard et al in Sweden: 1/300 babies born with effects of alcohol, 50% with FAS
10-20% of mental deficiency (IQ 50-80)
17% of cases of Cerebral Palsy
Studies in Seattle and Northern France show incidence >1/1000

7. Effects of alcohol on Fetus
We now know that ALCOHOL freely crosses the placenta
and is a TERATOGEN, causing
--brain damage
cerebellum, hippocampus,
basal ganglia, corpus callosum
--fetal alcohol syndrome
facial features, SGA, behavioral
and cognitive problems

8. Fetal Effects Increased cellular peroxidase activity
Decreased DNA synthesis
Disruption of protein synthesis
Impaired amino acid transport across placenta
Chronic fetal hypoxia from induced hypoglycemia

9. Effects of Alcohol

10. DEFINITIONS FAS : Growth impairment, facial dysmorphology
neurodevelopmental deficits & exposure
pFAS: Exposure, facial features and
neurodevelopmental deficits, but normal growth
ARBD: Exposure, normal growth and neurodevelopmental
functions but major dysmorphology
ARND: Exposure, normal facies and growth
but neurodevelopmental deficits
ND-PAE (will replace ARND) DSM 5th Ed.

11. Fetal alcohol syndrome
Growth restricted
Average IQ 63
Motor deficits
Tremulousness, irritability, hyperactive
Microcephaly,short palpebral fissures, long philtrum, thin upper lip, joint anomalies, small 5th fingernail, VSD/ASD

12. Fetal Alcohol syndrome
There is a narrow window of exposure, so that very few
infants have “classic” Fetal Alcohol syndrome
Most have “ Alcohol related neurodevelopmental disorder”
Other effects are: Esotropia (lazy eye)
: frequent ear infections
: partial or complete hearing loss
: average IQ FAS is 63
ARND with low IQ leads to issues with behavioral regulation,
impulsivity, social deficits, poor judgement, class disruption
ARND with “normal” IQ has learning disabilities, poor school
performance, poor “executive functioning” clumsiness, and
poor balance

13. Effects of Alcohol
Fetal alcohol syndrome is most severe effect, but only a small
proportion show the facies (1-3/1000)
Alcohol related neurodevelopmental disorder
(ARND) is much more prevalent (15-30/1000)
--exposure early in gestation causes not only features of FAS, but
brain anomalies
--exposure later does not have gross structural damage, but kills
nerve cells, inhibits synapses and myelin formation and biochemical
processes
Most common non-genetic cause of mental retardation

14. Effects of alcohol on Brain
Timing is important: First Trimester
--low set ears, cleft palate and lip, etc due to heavy exposure early in pregnancy
Midline structures such as LIMBIC SYSTEM affected
--information from sensory inputs processed and
sent to prefrontal cortex via Dopamine
--damage to hippocampus causes poor memory
--damage to corpus callosum causes “ODD”
Oppositional defiant disorder

16. Anomalies of corpus callosum
Begins at 39th day after conception
Adult morphology by 115th day
Damage early in 1st trimester
Partial or total agenesis
Poor motor coordination
Delayed milestones
Cognitive disabilities
Social difficulties

17. Stages of brain development

18. Effects of Alcohol on Brain
Third trimester exposure prevents MIGRATION and MYELINATION which causes LISSENCEPHALY

19. Effects of alcohol Severity of exposure
Greater exposure causes worse effects
--episodic binge drinking worse than same amounts consumed steadily
Chronicity of exposure:
--older mothers drinking for years cause higher risk for affected baby
--maybe they have reduced ability to metabolize alcohol

20. Prenatal alcohol exposure
In animals..
-- some interventions shown to ameliorate effects
--motor-training, physically and socially stimulating environment may improve outcomes
(brain plasticity)
-- better to protect from the insult than try to correct after the exposure

21. The developing brain Animal studies:
Rats reared in a complex environment had more synaptic connections, larger dendritic fields, more dendritic spines and more supportive glial tissue than those raised in barren cages.
Both early and late exposure were effective, but the sooner the exposure, the better.

22. Evaluating FASD using Zebrafish models

23. Zebrafish research
Danio rerio is a tropical fish from Ganges river and a great model
Easily raised (hundreds of eggs/week)
External fertilization so can observe development
Have nearly all organs of humans (except lungs, prostate, mammary glands
Have 84% of genes known to cause human disease
Can be easily exposed to ethanol during development and can look at key features of the human syndrome FASD

24. Zebrafish research in FASD
Exposing zebrafish embryos to ethanol can cause:
Increases in ubiquitinated proteins (mark proteins for degradation using the proteasome)
Upregulation of proteasome components
Specific inhibition of degradation of damaged or unnecessary proteins by the proteasome
May contribute to teratogenic effects this way

25. Zebrafish research in FASD

26. FASD research using Zebrafish
Can be used to model one of the alcohol- induced social deficits, which is a devastating symptom of FASD
Also work with cannabinoids has shown they are teratogenic early in pregnancy and can exacerbate the effects of alcohol
Studies show impairment of Sonic Hedgehog (Shh) signaling pathways and effects on craniofacial and brain development

27. Human Brain Development
Conception
Months
Years
AGE -6 -3 3 6 9 1 4 8 12 16
Sensing Pathways (vision, hearing)
Language
Higher Cognitive Function

28. Alcohol effects :Can we salvage the babies?
Primary goal of US DHHS is prevention
Central goal is to identify affected babies early and place in Early Intervention
May ameliorate effects on language and emotional dysregulation
May prevent academic, legal and psychiatric problems

29. EARLY DIAGNOSIS IS IMPORTANT!
Children with primary FAS/PAE have secondary disabilities later
which are potentially preventable
-- disrupted school experiences
-- mental health problems
-- inappropriate sexual behaviors
-- alcohol and drug abuse
-- incarceration and retention in the justice system
High prevalence of persistent psychiatric problems that
persist into adulthood
DIAGNOSIS BEFORE SIX YEARS OF AGE IS IDEAL

30. Challenges of Diagnosis
Maternal history is unreliable due to social stigmas
Different screening tools used :
AUDIT, CAGE, TWEAK, T-ACE, MAST, TLFB
Need screeners that are adequately trained to use these
Urine screening is inaccurate and therefore unreliable
Lack of clear physical findings in babies or children
Surveys of pediatricians reveal they feel incompetent
in making the diagnosis. Usually diagnose as ADHD
PHARMACOLOGIC INTERVENTIONS ARE DISASTROUS

31. Misdiagnosis and Missed Diagnosis
Chasnoff et al. Pediatrics vol.35#2Feb2015
Sample of 547 children :comprehensive multi-disciplinary and diagnostic evaluation
156 kids met criteria within fetal alcohol spectrum: 125 never diagnosed!
(80.1% missed diagnosis rate)
31 kids diagnosed as FAS..10 had diagnosis changed: (6.4% misdiagnosis rate)
21 kids remained with accurate diagnosis

32. Misdiagnosis and Missed Diagnosis
Chasnoff:
In this clinical sample, 86.5% of youth with FASD had never been diagnosed or were misdiagnosed
Clearly these high rates of inaccuracy have significant implications for intervention and therapeutic services.

33. WV Umbilical Cord Study, 2009
Source: Stitley, Michael, MD, et.al. “Prevalence of Drug Use in Pregnant West Virginia Patients,” West Virginia Medical Journal, Voll. 106, No. 4, 2010.

34. Results of Umbilical Cord Tissue Study, 2009

35. Detection of alcohol rates in pregnancy
Stitely ML. et al WV Med J 2010,106:48-52
--Cord study ( anonymous) 39/759 = 5.1%
--Birth cert/WV Birth score: 13/1079=1.2%
Detection rates at W&C Hosp, and WMC
--Cord study (anonymous) : 11/133 = 8.3%
--Birth cert/WV Birth score :2/283 =0.7%
Urine screens (3/1/12-1/31/13): 6/545 =1.1%

36. Detection of Prenatal Alcohol Exposure
Biological Markers of Alcohol Exposure in the Newborn:
Ethyl Glucuronide in Umbilical Cord Tissue
Fatty Acid Ethyl Esters in Meconium
Phosphatidylethanol in Blood 36

37. Ethyl Glucuronide
Ethanol is metabolized in the liver to produce EtG only when ethanol is present
EtG has been detected in urine, blood, sweat, hair and fingernails in adults and in newborn meconium and umbilical cord

38. Fatty Acid Ethyl Esters (FAEEs)
Fatty acid ethyl esters (FAEEs) are nonoxidative metabolites that are formed when alcohol conjugates to free fatty acids and fatty acyl- CoA. FAEEs have been detected in the umbilical cord tissue, blood, hair, and meconium of newborns.

39. Phosphatidylethanol (PEth)
Phosphatidylethanols (PEth) are a group of phospholipids with a common phosphoethanol head group onto which 2 fatty acid chains are attached.
Phosphoethanol head group
Fatty acid chains

40. PEth can form in red blood cells as a component of the cellular membrane.
PEth is a direct alcohol biomarker, meaning that ethanol is incorporated into the final product.

41. When Ethanol is not Present
Phosphatidylcholine (PC) PC
Phosphatidic acid and choline
Phospholipase D
(H2O)
Phosphatidylcholine is a major component of biological membranes, involved in membrane-mediated cell signaling and activation of other enzymes.
Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid, releasing choline into the cytosol. 41

42. When Ethanol is Present
Phosphatidylcholine (PC)
PEth PC
Phosphatidylethanol (PEth)
Phospholipase D
(Ethanol)
Phospholipase D has a higher affinity for ethanol than for water so when alcohol is present, Phospholipase D reacts preferentially with ethanol to convert phosphatidylcholine to phosphatidylethanol.
Human red blood cells do not have the enzymatic machinery to efficiently degrade PEth, causing the accumulation of PEth in the cellular membranes immediately following exposure to ethanol
The natural decomposition of PEth results in a slow elimination, with a half-life of approximately 4 days in adults - with PEth detectable in blood for up to 28 days following the last drinking episode 42

43. Detection of PEth in dried blood spots
US Drug testing Lab developed and validated a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for the extraction and detection of PEth from dried blood spots.
Filter paper cards lyse and fix red blood cells completely and prevent further PEth synthesis or the filter paper stabilizes PEth, minimizing sample degradation.
Many studies have examined PEth in blood and shown that it is a highly sensitive and specific indicator of alcohol use and misuse
Our laboratory developed and validated a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for the extraction and detection of PEth from DBS.
We have found that measurement of PEth in DBS samples is as reliable as detection in whole blood samples, and provides a more convenient technique for collection, transport, and storage.
The filter paper cards lyse and fix the red blood cells, preventing any further synthesis of PEth – even is ethanol is present in the blood sample.
The filter paper also stabilizes PEth, minimizing sample degradation when stored at RT and virtually eliminating any degradation at 4 or -20 degrees
The LC-MSMS method also alows us to set very low cut off – at 8 ng/mL 43

44. Validity of using DBS for detection of PEth
Baldwin et al (International Journal of Alcohol and Drug Research. 2015, 4(2), ) “Detection of PEth from newborn DBS cards can identify PAE and be used for retrospective surveillance of alcohol consumption during the last 3-4 weeks of pregnancy. Storage of cards at room temperature for three months had a 96.3% initial concentration detectable after 30 days” Bakhireva et al (Alcohol Clin Exp Res Vol 41, No pp ) Systematically estimated the prevalence of PAE in Texas by measuring PEth in 1000 infant residual dried blood spots from the Texas Newborn Screening Repository

45. Maternal Biomarker Studies
Study 1a:
Compare alcohol use in pregnancy rates based on self-report methods and analysis of urine ethanol screening and detection of alcohol biomarkers. 45

46. Results: Study 1a; maternal screening
314 patients screened for urine ethanol, self-report and PEth at the Charleston Area Medical Center 46

47. Improving screening for alcohol consumption during pregnancy with phosphatidylethanol (Bracero et al) Reproductive Toxicology 74(2017)
Compared rates of alcohol use between urine ethanol testing and self-reporting (Method 1) and PEth dried blood spot testing and self-reporting (Method 2)
Method 2 identified more alcohol users than Method 1.
Combining both methods resulted in a detection rate of 10.2% in the first trimester or at first antenatal visit

48. Purpose of Study 1b
To compare the ability of two alcohol biomarkers in identifying maternal alcohol consumption at the time of delivery.
Umbilical cord tissue Ethyl Glucuronide (EtG) vs Umbilical cord blood Phosphatidylethanol (PEth)
To determine the early neonatal outcomes associated with 3rd trimester maternal alcohol consumption during pregnancy by comparing umbilical cord PEth + vs. PEth - pregnancies

49. METHODS
Observational study of infants born to women who received prenatal care at WMC during
1/2013-8/2014
We compared the detection rate of umbilical cord blood PEth to the currently used Cordstat EtOH test (Ethyl Glucuronide Etg)

50. RESULTS 134 infants included in study
120 had u. cord tissue EtG testing for alcohol
120 negative
134 had u. cord PETH bloodspot testing for alcohol
101 negative
33 positive for alcohol(24.6%) Avg: ± [ ]ng/ml3

51. Comparisons of babies in both groups showed:
RESULTS
Comparisons of babies in both groups showed:
No significant differences in the following:
Gestational Age, Delivery Method, APGARS
Weight, Length and Head circumference
Neonatal morbidities (RDS, PDA, IVH, Sepsis etc)
No dysmorphology consistent with FAS

52. is not a reliable method of identification.
Conclusions
From these studies (and others) it appears that the best method of identifying babies at risk for developing effects of late prenatal alcohol exposure is the detection of PEth in dried blood spots from the umbilical cord at birth (or from dried blood spots NBS?)
It remains clear that dependence on physical parameters at birth (LBW, Facial features etc)
is not a reliable method of identification.

53. Screening for prenatal alcohol exposure (PAE) and corresponding short-term neonatal outcomes Maxwell et al, Reproductive Toxicology 85 (2019) 6-11
Compared a) PAE rates using the biomarker PEth in umbilical cord (UC) blood vs ethylglucuronide (EtG) in umbilical cord tissue b) pregnancy characteristics and neonatal outcomes in newborns positive vs negative biomarkers.
UC-PEth testing had a higher PAE detection rate (26%) vs UC tissue EtG (0%, p<0.01)
PAE (within 3-4 weeks of birth) was not associated with dysmorphic features or short-term adverse outcomes

54. Estimating Prenatal Alcohol Exposure in WV using residual newborn screening specimens
Estimate prevalence by screening dried blood spots for Peth
1800 DBS cards from state newborn screening repository in Nov, Dec 2017, and Jan 2018 from Office of Lab Services
Transferred to WV Birth Score office at WVU, Morgantown
Matched to unique birth score data, de-identified and sent to USDTL in Chicago for Peth analysis
1829 samples: 8.1% > 8 ng/ml. Range up to 346 ng/ml

55. PEth above the LOQ
148 samples
8.1%
PEth between the LOD
And the LOQ
375 samples
20.5%
PEth less than the LOD
1,307 samples
71.4%

56. Project WATCH matching data
Matched 1704 cards (92.3%)
136 > 8 ng/ml (8.0%)
Congenital abnormality 2/136 (1.4%)
IUSE 22/136 (16%)
NAS 7/136 (5.15%)

57. Project WATCH matching data
Tobacco use in pregnancy 47/136 (35%)
Gestational age >37 weeks 27/136 (19.8%)
Birthweight < 2500g 15/136 (11%)
g 88/136 (65%)
g 33/136 (24%)

58. Prenatal Alcohol Use by WV-SAMSHA Regions
(total n) N
% sample by region
Frequency for high PETH
Binomial proportion for high PETH
95% lower confidence interval
95% upper confidence interval
State
1471
112
7.61%
6.26%
8.97% 1 92
6.25 7
2.19%
13.03% 2
132
8.97 3
2.27%
0.00%
4.82%
152
10.3 26
17.11%
11.49%
24.05% 4
364
24.7 17
4.67%
2.50%
6.84% 5
424
28.8 27
6.37%
4.04%
8.69% 6
307
20.8 32
10.42%
7.01%
14.40%

59. Prenatal Alcohol Use and Maternal Characteristics
Statistically Significant p<0.05

60. Prenatal Alcohol Use and Infant Characteristics
Statistically Significant p<0.05

61. Alcohol Study data (N= 1729)
Insurance Coverage
Alcohol Study data (N= 1729)
Alcohol
IUSE
NAS
Smoking
Freq %
Private Insurance 
786
49.9% 62
48.8% 37
16.7% 12
17.1% 78
23.8%
Medicaid Insurance 
788
50.1% 65
51.2%
184
83.3% 58
82.9%
250
76.2%

62. Conclusions
From these studies (and others) it appears that the best method of identifying babies at risk for developing effects of late prenatal alcohol exposure is the detection of PEth in dried blood spots from the umbilical cord at birth or from dried blood spots collected for newborn screening.
It remains clear that dependence on physical parameters at birth (LBW, Facial features etc) is not a reliable method of identification.

63. Alcohol use in pregnancy in WV
A significant public health issue
Detection of PEth in DBS is an effective method for surveillance of prenatal alcohol exposure
Analysis in conjunction with Birth score data could identify babies at risk for developmental delay in early life

64. …stronger, healthier babies.64