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

1. New Frontiers in PathologyCase 2:Fetal Demise Associated WithInfluenza A Infection Richard W. Lieberman, M.D.Departments of PathologyandObstetrics & Gynecology

2. 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 Ultrasound Assessment • EGA by dates: 17+5 EGA by U/S: 15+6 • EGA by dates: 19+2 EFW < 3rd %ile • oligohydramnios and IUGR *MOM=multiple of the median Lieberman

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 Lieberman

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 Lieberman

5. Placenta Gross Lieberman

6. Histopathology Lieberman

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10. Fetopsy – GI Tract Lieberman

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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 Lieberman

13. 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 Lieberman

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15. Nucleus • electron hypodense areas • Cytoplasm • relatively uniform electron densities Lieberman

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18. Influenza A Virion Hx Nx: Influenza A serotyping Lieberman

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

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 *performed by Dr. Dafydd Thomas Lieberman

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22. Influenza A M1 cDNA Protein Lieberman

23. Fetal Lung Immunofluorescence Lieberman

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) Lieberman

25. 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 Pathology of Fetal Loss -- Classification Lieberman

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

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 Am J Obset Gynecol p. 433-44, May 2007Placenta 29:71-80, 2008 Lieberman

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

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: p747-756, 2006 Lieberman

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 Lieberman

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 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 Lieberman

32. 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 Lieberman

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 Lieberman

34. Extra Slides Lieberman

35. Abnormal Quad Screen & Outcome Lieberman

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