1. New Frontiers in Pathology Case 2: Fetal Demise Associated With Influenza A Infection
Richard W. Lieberman, M.D. Departments of Pathology and Obstetrics & Gynecology

2. Case Presentation
30 yo G1 presents with intrauterine fetal demise followed by induction of labor, and delivery of a 19+ weeks
technically 2nd trimester abortion
Antenatal Course:
Quad Screen at 14 weeks
MSAFP – 7 MOM*
inhibin A – 3.4 MOM
estradiol & hCG < 1 MOM
Two abnormal markers impart a 9 fold increase risk of fetal loss prior to 24 weeks.
Ultrasound Assessment
EGA by dates: EGA by U/S: 15+6
EGA by dates: EFW < 3rd %ile
oligohydramnios and IUGR
*MOM=multiple of the median
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3. Case Presentation (cont’d)
Additional Prenatal Labs
O positive
Rubella immune
Social History
healthcare worker
first trimester exposure to numerous patients with upper respiratory “flu”
asymptomatic(?)
1st trimester dizziness
influenza vaccine not yet available
Jan 3, 2008: EGA by dates: EGA by U/S: 15+6
Jan 14, 2008: EGA by dates: EFW < 3rd %ile
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4. Post-Partum Workup for Fetal Loss
TORCH Serologies
negative CMV and Toxoplasmosis
Thrombophilia
Protein S & C: normal
Factor V Leiden: negative
Chromosome Analysis: 46 XY
Pathology: Fetopsy & Placenta Evaluation
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5. Placenta Gross
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6. Histopathology
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10. Fetopsy – GI Tract
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12. Final Diagnosis Intrauterine fetal demise
second trimester spontaneous abortion
Diffuse villous fibrosis, perivillous and intravillous fibrin deposition
Focal trophoblastic hyperplasia
Chronic villitis and intervillositis (placentitis), histiocytic type
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13. Can we find a cause for this loss
Can we find a cause for this loss? What’s up with the sheets of histiocytes?
what else can be done?
Immunohistochemistry
very limited selection of antibodies
not cost effective to use multiple antibodies unless you have an idea of the causative pathogen
what about electron microscopy?
not readily available
primarily used in renal diagnostics
requires special expertise
operation AND analysis
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14. Identified area from slide
Identified area from slide. Paraffin gouge, thick section review, selecting for intervillous histiocytosis
Note the hypodensities along the nuclear membrane
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15. electron hypodense areas Cytoplasm
Nucleus
electron hypodense areas
Cytoplasm
relatively uniform electron densities
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18. Influenza A Virion
Hx Nx: Influenza A serotyping
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19. * *http://www.nimr.mrc.ac.uk/elecmicroscopy/examples/staining/
The typical EM takes cultured or ground fresh tissue and ultrcentrifuges. Formalin fixation does have an effect on the appearance, hence they are not easily found.
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20. Confirmation of Influenza A
immunofluorescence*
antibody to Inf A H1N1 (USSR)
Antibody specific to H1N1 viral protein
dual staining
Keratin mix [FITC green]
Viral antibody [Cy5 red]
RT-PCR*
Total RNA extracted from formalin fixed paraffin embedded tissue
RT reaction performed with random decamers
primers specific for M1 cDNA & GAPDH
GAPDH housekeeping gene positive in all cells
Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is an important enzyme in glycolysis ... GADPH is responsible for catalyzing the reversible conversion
*performed by Dr. Dafydd Thomas
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21. Lieberman H1N1: 69% A/Solomon Isles/3/2006 (In 2007 Vaccine)
H1N1: 24% A/Brisbane/59/ (Not In Vaccine)
H3N2: 22% A/Wisconsin/67/2005 (In 2007 Vaccine)
H3N2: 71% A/Brisbane/10/2007 (In Southern Hemisphere Vaccine Only)
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22. Influenza A M1 cDNA Protein
Four placental blocks from this case. RNA extracted from all four blocks with positive and negative controls
Pos ctrl: respiratory cells commercially available with Inf A & B. Inf A extracted from control
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23. Fetal Lung Immunofluorescence
antibody to Inf A H1N1 (USSR)
Antibody specific to H1N1 viral protein
dual staining
Keratin mix [FITC green]
Viral antibody [Cy5 red]
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24. Final Diagnosis Intrauterine fetal demise
second trimester spontaneous abortion
Diffuse villous fibrosis, perivillous and intravillous fibrin deposition
Chronic villitis and intervillositis (placentitis), histiocytic type.
Influenza A virus infection (H1N1) with ultrastructural, immunohistochemical, and PCR confirmation
transplacental passage of Influenza A (H1N1) to fetus
placental: intravillous (hematogenous or direct)
intra-amnionic: ingested and inhaled (surface epithelial positive)
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25. Pathology of Fetal Loss -- Classification
pre-placental (maternal)
maternal vascular obstruction/disruption
developmental
implantation site
inflammatory
mixed
placental (maternal-fetal interface)
fetal vascular
obstruction/disruption
post-placental (fetal)
fetal inflammatory response
meconium
cord related
congenital infection
hydrops fetalis
developmental
anatomic
chromosomal
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26. Stillbirth or Abortion?
stillbirth: “delivery of an infant with no sign of life between 20 weeks gestation and term”
perinatal loss*
before 20 weeks ~15 per 1000 live births
between 20 weeks & term ~6 per 1000 live births
Our Case: Best EGA = 19+2 weeks
initial discrepencies between the pathology requisition and the medical record were resolved after careful chart review. Best EGA was 19+2 weeks gestation
Rate is per 1000 live births
*Centers for Disease Control and Prevention. National Center for Health Statistics. VitalStats. (2003-4)
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27. Causes of Perinatal Death
Following fetopsy, placenta path & record review:
unresolved: ~50%
cord related: 5-28%
infectious: 10-25%
more likely at early GA
vasculopathy: ~15%
fetal-maternal hemorrhage: 3-14%
genetic: 6-12%
Problem: No placental evaluation in 10-44% of all intrapartum deaths
Unresolved cases may also include Maternal Conditions like advanced maternal age, or adolescent pregnancy
Am J Obset Gynecol p , May 2007 Placenta 29:71-80, 2008
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28. Placental Chronic Villitis
Etiologies
undetermined – 90%
TORCH
toxoplasmosis
other = parvovirus b19
rubella
cytomegalovirus
herpesvirus
varicella
Enterovirus (Coxsackie)
EBV
… and Influenza(?)
VUE – villitis of undetermined etiology
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29. Placental Viral Infections: Australian Study
Multiplex PCR of 105 Placentas
CMV, Parvo, Human Herpes Virus (HHV), mycoplasma & ureaplasma
Low Risk Group (asymptomatic)
CMV 4%, Parvo 1%, Ureaplasma 1%
High Risk Group (preg. loss or seroconversion)
CMV 64%, HHV 9%
Histological changes only in high risk group
J Med Virol 78: p , 2006
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30. Ultrastructural Analysis of Chronic Villitis
Rarely Studied
Ireland: EM of VUE (n=34)
41 % with viral particles (c/w rotavirus, coronavirus, HPV, enterovirus and adenovirus)
so far, abstract only (Placenta 26: A38, 2005)
Parvovirus B19
used fresh tissue and immune EM (2 & 6)
38 cases of parvo, 8 cases with ultrastructural evaluation
increased size of viral particles with formalin fixation
Scattered case reports of rubella, hepatitis, RSV…
No mention of Influenza virus
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31. Influenza Virus Infection in Pregnancy
Seasonal Influenza A or B
11-25% of pregnant women affected
“dearth of accurate information regarding the biological consequences of maternal virus infection”
Br J Obstet Gynecol 107: p , 2000
Influenza Outbreak Stats ( )
association with increase in maternal mortality
ACOG recommends Influenza Vaccine for ALL pregnant women
no association with  stillbirth, neonatal death, or malformation
observation of possible  rate of miscarriage in first trimester
H1N1: 69% A/Solomon Isles/3/2006 (In 2007 Vaccine)
H1N1: 24% A/Brisbane/59/ (Not In Vaccine)
H3N2: 22% A/Wisconsin/67/2005 (In 2007 Vaccine)
H3N2: 71% A/Brisbane/10/2007 (In Souther Hemisphere Vaccine Only)
Public Health Reports 78(1): 1-11, 1963
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32. Transplacental Passage of Influenza?
Br J Obstet Gyencol 107: p , 2000
2nd and 3rd trimester exposure common
significant increase in complications, but no “specific” complication
no evidence of transplacental passage
Am J Obstet Gynecol 149(8):p856-9, 1984
case report: viral particles identified in amniocentesis fluid with seroconversion of mother & baby
Arkh Patol (Russian abstract) 49(9):p19, 1987
Influenza A/B virus antigen in 32 of 186 placentas
immunofluorescence and light microscopy
noted in trophoblast, decidual cells and villous endothelium
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33. Case 2 Summary
First report: mid-trimester fetal loss (abortion) associated with Influenza A (H1N1)
Q: cause-effect? … compelling time-course with 1st trimester exposure
Novel use of Electron Microscopy
analysis of histiocytic proliferation and identification of budding fully packaged viruses
morphology consistent with Influenza virus
directed selection of pathogen for confirmatory testing
not practical for routing testing
Immunofluorescence and PCR
confirmation transplacental passage of virus by identifying capsid protein in both the maternal and fetal space
The Future?
multiplex PCR or DNA microarray “chip”
primers for “common” pathogens effecting pregnancy and placental function
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34. Extra Slides
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35. Abnormal Quad Screen & Outcome
Two abnormal markers impart a 9 fold increase risk of fetal loss prior to 24 weeks.
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