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New Frontiers in PathologyCase 2:
Fetal Demise Associated WithInfluenza A Infection
Richard W. Lieberman, M.D.Departments of Pathology
andObstetrics & Gynecology
New Frontiers: 2008 - 2Lieberman
Case Presentation
• 30 yo G1 presents with intrauterine fetal demise followed by induction of labor, and delivery of a stillborn @ 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
*MOM=multiple of the median
1. EGA by dates: 17+5 EGA by U/S: 15+6
2. EGA by dates: 19+2 EFW < 3rd %ileoligohydramnios and IUGR
Ultrasound Assessment
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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
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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?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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Nucleus•electron hypodense areas
Cytoplasm•relatively uniform electron densities
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Influenza A Virion
Hx Nx: Influenza A serotyping
New Frontiers: 2008 - 19Lieberman*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 = 19+2 weeks*Centers for Disease Control and Prevention.National Center for Health Statistics. VitalStats. http://www.cdc.gov/nchs/vitalstats.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 29:71-80, 2008
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Placental Chronic Villitis
• chronic villitis • Etiologiesundetermined – 90%undetermined – 90% – TORCH
• toxoplasmosis• other = parvovirus b19• rubella• cytomegalovirus• herpesvirus• varicella• Enterovirus (Coxsackie)• EBV• … and Influenza(?)
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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: p747-756, 2006
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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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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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