New Frontiers in Pathology Case 2: Fetal Demise Associated With Influenza A Infection

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New Frontiers in PathologyCase 2Fetal Demise
Associated WithInfluenza A Infection

• Richard W. Lieberman, M.D.Departments of
  PathologyandObstetrics Gynecology

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Case Presentation

• 30 yo G1 presents with intrauterine fetal demise
  followed by induction of labor, and delivery of a
  stillborn _at_ 19 weeks
• technically 2nd trimester abortion
• Antenatal Course
• Quad Screen at 14 weeks
• MSAFP 7 MOM
• inhibin A 3.4 MOM
• estradiol hCG lt 1 MOM

Ultrasound Assessment

• EGA by dates 175 EGA by U/S 156
• EGA by dates 192 EFW lt 3rd ile
• oligohydramnios and IUGR

MOMmultiple of the median
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Case Presentation (contd)

• 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

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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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Placenta Gross
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Histopathology
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Fetopsy GI Tract
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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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Can we find a cause for this loss?Whats 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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• Nucleus
• electron hypodense areas
• Cytoplasm
• relatively uniform electron densities

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Influenza A Virion
Hx Nx Influenza A serotyping
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http//www.nimr.mrc.ac.uk/elecmicroscopy/examples
/staining/
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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

performed by Dr. Dafydd Thomas
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Influenza A M1 cDNA Protein
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Fetal Lung Immunofluorescence
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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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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
• developmental

• post-placental (fetal)
• fetal inflammatory response
• meconium
• cord related
• congenital infection
• hydrops fetalis
• developmental
• anatomic
• chromosomal

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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 192 weeks

Centers for Disease Control and
Prevention.National Center for Health
Statistics. VitalStats. http//www.cdc.gov/nchs/v
italstats.htm (2003-4)
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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

Am J Obset Gynecol p. 433-44, May 2007Placenta
2971-80, 2008
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Placental Chronic Villitis

• chronic villitis

• Etiologies
• undetermined 90
• TORCH
• toxoplasmosis
• other parvovirus b19
• rubella
• cytomegalovirus
• herpesvirus
• varicella
• Enterovirus (Coxsackie)
• EBV
• and Influenza(?)

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Placental Viral InfectionsAustralian 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 p747-756, 2006
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Ultrastructural Analysis of Chronic Villitis

• Rarely Studied
• Ireland EM of VUE (n34)
• 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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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 1282-9, 2000
• Influenza Outbreak Stats (1957- 61)
• 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

Public Health Reports 78(1) 1-11, 1963
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Transplacental Passage of Influenza?

• Br J Obstet Gyencol 107 p 1282-9, 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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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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Extra Slides
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Abnormal Quad Screen Outcome
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