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

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


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


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


Placenta Gross


Histopathology





Fetopsy – GI Tract



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


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



Nucleus•electron hypodense areas

Cytoplasm•relatively uniform electron densities




Influenza A Virion

Hx Nx: Influenza A serotyping

New Frontiers: 2008 - 19Lieberman*http://www.nimr.mrc.ac.uk/elecmicroscopy/examples/staining/

*


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



Influenza A M1 cDNA Protein


Fetal Lung Immunofluorescence


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)


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


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)

http://www.cdc.gov/nchs/vitalstats.htm


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


Placental Chronic Villitis

• chronic villitis • Etiologiesundetermined – 90%undetermined – 90% – TORCH

• toxoplasmosis• other = parvovirus b19• rubella• cytomegalovirus• herpesvirus• varicella• Enterovirus (Coxsackie)• EBV• … and Influenza(?)


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


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


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


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


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


Extra Slides


Abnormal Quad Screen & Outcome





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

Documents

lieberman slide

new frontiers

lieberman histopathology

lieberman influenza

lieberman placenta gross

analysis slide

available slide

lieberman stillbirth