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

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

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 New Frontiers: Lieberman Case Presentation 30 yo G1 presents with intrauterine fetal demise followed by induction of labor, and delivery of a 19+ weeks –technically 2 nd 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: EGA by dates: 19+2 EFW < 3rd %ile oligohydramnios and IUGR Ultrasound Assessment

3 New Frontiers: Lieberman 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(?) –1 st trimester dizziness –influenza vaccine not yet available

4 New Frontiers: Lieberman 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

5 New Frontiers: Lieberman Placenta Gross

6 New Frontiers: Lieberman Histopathology

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10 New Frontiers: Lieberman Fetopsy – GI Tract

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12 New Frontiers: Lieberman 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

13 New Frontiers: Lieberman 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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15 New Frontiers: Lieberman Nucleus electron hypodense areas Cytoplasm relatively uniform electron densities

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18 New Frontiers: Lieberman Influenza A Virion Hx Nx: Influenza A serotyping

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

20 New Frontiers: Lieberman 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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22 New Frontiers: Lieberman Influenza A M1 cDNA Protein

23 New Frontiers: Lieberman Fetal Lung Immunofluorescence

24 New Frontiers: Lieberman 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)

25 New Frontiers: Lieberman 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

26 New Frontiers: Lieberman 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. (2003-4)http://www.cdc.gov/nchs/vitalstats.htm

27 New Frontiers: Lieberman 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 , May 2007 Placenta 29:71-80, 2008

28 New Frontiers: Lieberman Placental Chronic Villitis chronic villitisEtiologies undetermined – 90% undetermined – 90% –TORCH toxoplasmosis other = parvovirus b19 rubella cytomegalovirus herpesvirus varicella Enterovirus (Coxsackie) EBV … and Influenza(?)

29 New Frontiers: Lieberman 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

30 New Frontiers: Lieberman 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

31 New Frontiers: Lieberman 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 Public Health Reports 78(1): 1-11, 1963

32 New Frontiers: Lieberman Transplacental Passage of Influenza? Br J Obstet Gyencol 107: p , 2000 –2 nd and 3 rd 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

33 New Frontiers: Lieberman Case 2 Summary First report: mid-trimester fetal loss (abortion) associated with Influenza A (H1N1) –Q: cause-effect? … compelling time-course with 1 st 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

34 New Frontiers: Lieberman Extra Slides

35 New Frontiers: Lieberman Abnormal Quad Screen & Outcome

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