Evaluation of StillbirthKaty Kemnetz, PGY2
OBJECTIVESEvaluation of stillbirth
Objectives
The participant will be able to:Identify the conditions that have been best
demonstrated to cause stillbirthEvaluate a stillbirth using the most effective
workupAccurately formulate an etiology for a
stillbirth, when possibleEmploy the recmomended hospital policies
for management of stillbirth
OutlineDefinitionsCauses of stillbirthWorkup of stillbirthOther considerationsConclusions
DefinitionsEvaluation of Stillbirth
DefinitionsSpontaneous abortion Intrauterine fetal demiseMiscarriage<20 weeks gestation or <500g
“Stillbirth” >20 weeks gestation or >350g—state
dependent◦ 350g is 50%ile for 20 weeks gestation
◦ Illinois: >20 weeks gestation
“Delivery of a fetus showing no signs of life as indicated by the absence of breathing, heart beats, pulsation of the umbilical cord, or definite movements of voluntary muscles”
Does not include terminations of pregnancy or IOL for previable PPROM
CausesEvaluation of stillbirth
Causes of stillbirth>30 classification systems existImportant to distinguish between
◦Underlying cause of death◦Mechanism of death◦Risk factors
CLASSIFICATION OF STILLBIRTH
The National Institute of Child Health and Human Development
Classification of StillbirthEunice Kennedy Shriver workshop 2007National Institute of Child Health and Human
Development“An optimal classification system would
identify the pathophysiological entity initiating the chain of events that irreversibly lead to death”
Criteria for “cause”Epidemiologic data demonstrate an excess of stillbirth
associated with that conditionBiologic plausibility that the condition causes stillbirthEither rarely seen in association with live births or,
when seen in live births, results in a significant increase in neonatal death
A dose-response relationship exists◦ The greater the “dose” of the condition, the greater the risk of
fetal deathAssociated with evidence of fetal compromiseThe stillbirth likely would not have occurred if that
condition had not been present
Causes of stillbirth
Reddy, UM et al. “Stillbirth Classification—Developing an International Consensus for Research.” Obstetrics and Gynecology, Vol 114, No 4, October 2009.
INFECTIONSCauses of stillbirth—NICHHD workshop consensus
InfectionAssociated with 10-20% of stillbirths in
developed countriesHigher association with preterm birthSometimes difficult to prove causality
InfectionAscending infectionamniotic fluid or
fetusfetal pneumonitisHematogenous spreadvillitis
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
Mechanism of fetal deathSevere maternal
illnessPlacental infection
that prevents oxygen/ nutrients from crossing to the fetus
Fetal infection that causes a lethal congenital deformity
Fetal infection that damages a vital organ
Precipitation of preterm labor, with intrapartum fetal death
Infections must be provenSigns of infection in the fetus
◦Evidence on autopsy of extensive organ involvement
Positive fetal culturesPositive maternal cultures plus
chorioamnionitis/ funisitis
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
Causes of IUFD: Spirochetes
Organism Maternal disease Comment
Treponema palladium Syphilis Major cause of stillbirth when maternal prevalence is high
Borrelia burgdorferi Lyme disease Tick borne; not a common cause of stillbirth
Borrelia recurrentis Relapsing fever Tick borne; common in the Western US; rare cause of stillbirth
Borrelia duttonii Relapsing fever Tick borne; sub-Saharan Africa; important cause of stillbirth
Leptospira interrogans Leptospirosis Uncommon
Severe placental
dysfunction
ProtozoaOrganism Maternal disease Comment
Trypanosoma brucei Trypanasomiasis Tsetse fly
Trypanosoma cruzi Chagas disease Kissing bug
Plasmodium falciparum Malaria Common in endemic areas
Plasmodium vivax Malaria Mosquitoes
Toxoplosmosis gondii Toxoplasmosis Rare
Severe placental
dysfunction
VirusesOrganism Maternal disease Comment
Parvovirus B19 Erythema infectiosum Likely the most common viral etiologic agent
Coxsackie A and B Various May be important
Echovirus Various Importance unknown
Enterovirus Various Importance unknown
Hepatits E virus Fulminant hepatic failure Especially in endemic areas
Poliovirus Polio Historic cause
Varicella zoster Chickenpox Rare cause
Rubella German measles Rare in developed countries
Mumps Parotitis Rare in developed countries
Rubeola Measles Rare in developed countries
Cytomegalovirus Asymptomatic Case reports
HIV AIDS Not likely causative
Influenza Respiratory tract infection Severe maternal illness
Causes lethal fetal
anomalies
BacteriaOrganism Maternal disease Comment
E. Coli Asymptomatic Probably the most common organism assoc with stillbirth
GBS Asymptomatic Common cause of stillbirth
Klebsiella Asymptomatic Common cause of stillbirth
Enterococcus Asymptomatic
Ureaplasma, mycoplasma Asymptomatic
Listeria monocytogenes Listeriosis Transmitted transplacentally
Chlamydia trachomatis Pelvic infection Suggested cause—case reports
Neisseria gonorrhoeae Pelvic infection Suggested cause—case reports
Candida albicans Thrush; vaginitis Confirmed in case reportsGoldenberg RL, Thompson C. The infectious origins of stillbirth. Am J Obstet Gynecol2003;189:861–73
Severe placental
dysfunction
Damage to vital organs
(brain, heart)
MATERNAL MEDICAL CONDITIONS
Causes of stillbirth—NICHHD workshop consensus
Simpson, LL. Maternal medical disease: Risk of Antepartum Fetal Death. Semin Perinatol, 2002, 26, 47.
Condition Estimated stillbirth rate per 1000 births in patients with the condition
All pregnancies 6-7
Chronic hypertension 5-25
Superimposed preeclampsia 52
Gestational hypertension and mild preeclampsia
9
Severe preeclampsia 21
Eclampsia 18-48
HELLP syndrome 51
Hypertensive disorders
Hypertensive disordersMechanism of fetal demise:
To consider cause of death:
Placental insufficiency◦ IUGR
◦ Abruption
If it progresses to eclampsia If it is associated with
placental abruption or fetal growth restriction
Simpson, LL. Maternal medical disease: Risk of Antepartum Fetal Death. Semin Perinatol, 2002, 26, 47.
Condition Estimated stillbirth rate per 1000 births in patients with the condition
All pregnancies 6-7
Gestational diabetes 5-10
Type 1 diabetes 6-10
Type 2 diabetes 35
Diabetes
DiabetesMechanism of fetal demise:
To consider cause of death:
Congenital abnormalityPlacental dysfunctionObstructed labor and
intrapartum deathMacrosomia
◦ Fetal hyperglycemiafetal insulin productionexcessive fetal growthmetabolic acidosis
Signs of intrauterine or intrapartum asphyxia
LGA fetusSGA fetusSevere malformationPlacenta demonstrates
characteristic histologic findings◦ Large edematous villi
◦ Increased prominence of cytotrophoblasts
Simpson, LL. Maternal medical disease: Risk of Antepartum Fetal Death. Semin Perinatol, 2002, 26, 47.
Condition Estimated stillbirth rate per 1000 births in patients with the condition
All pregnancies 6-7
Stable treated hyperthyroidism 0-36
Uncontrolled thyrotoxicosis 100-156
Subclinical hypothyroidism 0-15
Overt hypothyrodism 15-125
SLE 40-150
Mild chronic renal disease 15
Moderate and severe chronic renal disease 32-200
Cholestasis of pregnancy 12-30
Thyroid/renal disorders
Thyroid/renal disordersThyroid disorders Renal disordersGraves disease, where thyroid-
stimulating hormone receptor antibody causes fetal toxicosis
Untreated thyroid disorders
Linear relationship between maternal creatinine and risk of fetal demise
Systemic Lupus ErythematosusStillbirth rates are higher in the presence of HTN, nephritis, or APLCirculating auto-antibodies, anti-Ro, anti-La
Congenital heart block, hydrops
Maternal medical conditionsRisk is a continuum
Reddy, UM et al, 2009
THROMBOPHILIASCauses of stillbirth—NICHHD workshop consensus
ThrombophiliasAntiphospholipid syndrome Inherited thrombophilias Inflammation, thrombosis, and
infarction in the placentaClear histopathological or
clinical evidence of placental insufficiency
Factor V Leiden mutation, antithrombin III deficincy, prothrombin gene mutation, protein C deficiency, protein S deficiency
Placental infarction and thrombosis
Two large prospective cohort studies found no association between factor V Leiden mutation and pregnancy loss or placental insufficiency
Thrombophilias are common in healthy women with normal outcomes
Thrombophilias should only be considered as the cause of stillbirth with:• Evidence of placental
insufficiency such as fetal growth restriction or infarction and • Recurrent fetal loss
ALLOIMMUNIZATIONCauses of stillbirth—NICHHD workshop consensus
AlloimmunizationRed cell alloimmunization Platelet alloimmunization
Anti-Rhesus D, anti-Rhesus C, anti-Kell
Must have a positive indirect Coombs test
Antibody titers more than 1:16 (or 1:8 for anti-Kell)
Evidence of fetal anemia with hydrops
Evidence of fetal extramedullary hematopoeisis
HPA-1a, HPA-5a, HPA-4Maternal antibodies against
paternal and fetal platelet antigens
Parental platelet incompatibility for the pertinent antigen
Fetal thrombocytopeniaMassive intracranial
hemorrhage
CONGENITAL MALFORMATIONSCHROMOSOMAL ABNORMALITIES
Causes of stillbirth—NICHHD workshop consensus
CriteriaEpidemiologic data demonstrating an excess
of intrauterine mortalitySeen rarely in liveborn neonatesWhen seen in liveborn neonates, it
frequently results in neonatal deathBiologic plausibility that it can result in
death
Cause of death Stillbirth cases, % (out of total 2211 assessed)
Trisomy 21 1.53
Jugulolymphatic obstruction 1.45
Turner syndrome 1.09
Twin-twin transfusion syndrome 1.09
Anencephaly 1.0
Trisomy 18 0.81
Amnion disruption sequence 0.59
Lower mesodermal defects 0.50
Idiopathic nonimmune fetal hydrops 0.50
Trisomy 13 0.41
Congenital malformations
Reddy, UM et al. “Stillbirth Classification—Developing an International Consensus for Research.” Obstetrics and Gynecology, Vol 114, No 4, October 2009.
Chromosomal abnormalitiesIncidence
Cytogenetic abnormalities account for 6-13% of all stillbirths
This may be higher because 40-50% attempted karyotypes fail to grow
23% monosomy X, 23% trisomy 21, 21% trisomy 18, 8% trisomy 13
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
Chromosomal abnormalitiesAutosomal recessive disorders
Autosomal dominant disorders
Alpha thalessemia, Smith Lemli Opitz
Phenotype in lethal cases may differ from live cases
Skeletal dysplasiasMore often spontaneous
mutations
Firestein: Kelley’s Textbook of Rheumatology, 9th ed. Saunders 2012
FETOMATERNAL HEMORRHAGE
Causes of stillbirth—NICHHD workshop consensus
Fetomaternal hemorrhageThe cause 4% of all stillbirthsRisk factors:
◦Placental abruption◦Abdominal trauma◦Multiple gestation◦Abnormal fetal testing
Fetomaternal hemorrhageRisk of stillbirth depends on
◦Amount of hemorrhage◦Acute/chronic◦Gestational age
A threshold of 20 mL/kg of fetal bleeding is associated with increased risk of stillbirth
Autopsy confirmation of fetal anemia and hypoxia
PLACENTAL CAUSESCauses of stillbirth—NICHHD workshop consensus
Placental causesPlacenta previa, vasa
previa, neoplasmsPlacental abruption
has 8.9 relative risk of stillbirth◦May be considered the
cause of death if >30% of the placenta shows signs of abruption Reddy, UM et al, 2009
Placental causesAny disease that causes an SGA placenta
may result in stillbirth◦<5% expected weight for gestational age◦Preeclampsia, DM, HTN, renal, chronic infections
Any disease that causes an LGA placenta may result in stillbirth◦>95% expected weight for gestational age◦Hydrops fetalis, DM, syphilis
UMBILICAL CORD PATHOLOGY
Stillbirth classification—NICHHD workshop consensus
Umbilical cord pathologyAccount for 3.4-15% of stillbirthsVelamentous insertion
◦If it leads to a vasa previa or bleeding during laborUmbilical cord prolapse
◦Associated with prematurity, malpresentation, mutiparity, obstetric manipulation
Umbilical cord occlusion◦Cord prolapse, entanglement (mono-mono twins)◦Torsion◦Rupture, strictures, hematomas
Velamentous insertion
Umbilical cord pathologyNuchal cord
◦Occurs in up to 30% of normal pregnancies◦Not associated with an increased risk of stillbirth in
study of 14,000 deliveriesTrue knot
◦Also common in live births◦Grooving of the cord, constriction of the umbilical
vessels, edema, congestion, thrombosisrequired to claim it is the etiology
Isolated finding of a nuchal cord or a true knot at the time of
delivery is insufficient evidence that cord
accident is the cause of stillbirth
•Exclude other relevant causes of stillbirth•Find evidence of hypoxia and cord occlusion on postmortem examination
MULTIFETAL GESTATIONStillbirth classification—NICHHD workshop consensus
Complications of multifetal gestationMonochorionic
placentation◦Twin-twin
transfusion syndrome occurs in 9% of mono-di twins
◦Mortality can be 90% in untreated cases
Complications of multifetal gestationMono-mono twins
◦Cord entanglement, preterm birth, growth impairment, malformations, genetic abnormalities, vascular anastomoses
Gabbe: Obstetrics: normal and problem pregnancies, 6th ed. Saunders 2012
UTERINE COMPLICATIONSCauses of stillbirth—NICHHD workshop consensus
Uterine complicationsUterine rupture
◦Evidence of obstructed circulationUterine abnormalities
◦There is an increased risk of uterine abnormalities in women with recurrent pregnancy loss/stillbirth
◦Possibly due to poorly vascularized uterine tissue or space constraints
◦ Increased risk of PPROM, cervical insufficiency, preterm labor
◦Septate uterus has highest risk of stillbirth and placental abruption
WorkupEvaluation of stillbirth
IMPORTANCEWorkup of stillbirth
Importance of a stillbirth evaluationCounseling for risk of recurrencePossible intervention to reduce recurrence
riskFacilitate emotional closure and healing
Most stillbirths remain unexplainedIncomplete evaluation
◦Lack of clinician awareness◦Concerns of the family
Lack of single universally accepted classification scheme◦Difficult to assign a definitive cause
Unknown cause◦Sometimes despite thorough evaluation
OVERVIEWWorkup of stillbirth
OverviewRecommended studies Sometimes helpful Not generally useful
•Autopsy•Placental pathology•Karyotype•Kliehauer-Betke•Indirect Coombs•Acquired thrombophilia panel•Anti B2-glycoprotein ab•Toxicology screen
•Syphilis serology•Inherited thrombophilia panel•Glucose screening•TSH•CMV, toxoplasmosis, other infectious•Bile acids•Sonohysterogram
•Routine TORCH titers•ANA testing•Cultures of placental membranes
Silver et al, 2010
How do we know what works? “Evaluation of 1025 fetal deaths: proposed diagnostic
workup.” Korteweg FJ, Erwich JJHM, Timmer A, et al. AJOG 2012. Prospective cohort study in 50 Dutch hospitals Singleton IUFDs at >=20wks gestation, excluding pregnancy
terminations and intrapartum deaths Cause of death determined by a multidisciplinary panel (2
obstetricians, an OB resident, perinatal pathologist, neonatologist, genetics, microbiologist)◦ Initial demonstratable pathophysiological entity initiating the chain
of events that irreversibly led to death Contribution of each diagnostic test for determination of cause of
death was determined by the panel◦ Valuable for “establishing cause of death” or “excluding cause of
death”
MEDICAL HISTORYWorkup of stillbirth
Thorough medical/obstetrical history
Pregnancy complications
Maternal medical history
Genetic abnorm
alities
Substance usePregnancy history
FAM
ILY H
ISTO
RY
EXAMINATIONOF THE FETUSAND PLACENTA
Workup of stillbirth
EXAMINATION OF THE FETUSWorkup of stillbirth
Physician’s examWeight, head circumference, lengthPhotographs
◦Frontal and profie◦Whole body, face, extremeties, palms,
abnormalities
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
Finding Time of demise
Brown or red discoloration of the cord stumpDesquamation >1cm
>6 hours ago
Desquamation of face, back, abdomen >12 hours ago
Desquamation >5% of the body or >2 body zones
>18 hours ago
Skin color brown or tan >24 hours ago
Mummification(reduced soft tissue, leathery skin, dark brown)
>2 weeks ago
Time of demise
“Estimating the time of death in stillborn fetuses: III. External fetal examination; a study of 86 stillborns.” Genest DR, Singer DB. Obstet and Gynecol, 1992; 80(4), 593
AutopsyNew information that influences counseling in
26-51% of casesDutch study: valuable in 72% of casesHowever, it is performed in <50% of cases
◦Clinician hesitation to recommend autopsy◦Patient reservations
AutopsyAsk about patient reservationsEmphasize potential benefits to familyAllow the family ample time to hold the baby
and perform religious/cultural activities first
AutopsyMedical autopsies require consent of next-of-
kinConsent should be explicit enough that
parents can limit the autopsy procedure
Illinois Masonic Policy“An autopsy is an option for fetuses 20 weeks
or greater, but if clinically indicated or requested by the family, an autopsy can be offered before 20 weeks.”
If an autopsy is not requested, a Surgical Pathology examination can be performed
Components of an autopsyReview of the clinical history, laboratories,
antenatal imagingExternal examination:
◦Weight, crown-rump length, foot length, crown-heel length, occipital-frontal circumference
◦ Joint mobility, facial features, hands, feet, genitaliaIntraoral examination:
◦Pharynx, soft palate, uvulaTake into consideration changes from macerationPhotographs
External examination
Lentz: Comprehensive Gynecology, 6th ed
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
Components of an autopsyInternal examination
◦Y or T shaped incision on anterior trunk◦Organs examined in situ◦Heart sometimes opened in situ, sometimes
removed and fixed Vascular connections, septal defects
◦Organs removed in bloc for detailed dissection◦Spinal cord removed
Components of an autopsyBrain
◦Coronal scalp incision extending to behind the ears◦Skull is opened along cranial sutures◦Can extend into posterior neck for posterior fossa
(Dandy-Walker, Chiari) by removing occipital bone and cervical vertebrae
◦Brain is placed in formalin for 10 days before examination
BrainChiari malformation—downward displacement of medulla
Fligner 2011
Components of an autopsySamples of tissue for
◦Microscopic evaluation◦Viral cultures◦Bacterial cultures
Metabolic studies (Guthrie card)
Histologic exam
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
Extramedullary hematopoeisis in fetal liver in fetus with non-immune hydrops
What autopsy can identifyMarkers of IUGR
◦Head circumference percentile higher than weight percentile
◦Brain weight appropriate for gestation while other organs are appropriate for body weight
◦Altered brain weight:liver weightKidneys
◦Polycystic kidney diseaseCertain brain pathologiesCongenital anomalies
Kidneys
A: 28 wks, suspicious for autosomal recessive polycystic kidney diseaseB: confirmed on autopsyC: 22 wks, suspicious for ARPKDD: Autopsy showed diffuse renal cystic dysplasia
Autopsy can provide evidenceInfectionAnemiaHypoxia
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
Alternatives to autopsyPartial autopsy
◦Head-sparing autopsy (may miss CNS pathology)External examination by a trained pathologist
◦Can identify syndromes, congenital anomalies, timing of death, growth anomalies
◦Will likely miss fetal infections and internal anomalies
External examination with selected biopsies◦More likely to identify fetal infections
Alternatives to autopsy: MRI
◦Very good for CNS pathology Sometimes better than
autopsy, because fetal brain has high water content and liquefies
◦Fluid collections and effusions in the body
◦May miss cardiac anomalies, bowel anomalies
◦Cannot diagnose infections or metabolic disease
Advantages Disadvantages
MRI
24 wks, massive bilateral intraventricular hemorrhage
Fligner 2011
MRIImage-guided percutaneous biopsies
◦<50% success rate at obtaining tissue in one study◦Somewhat inferior to autopsy:
12 cases where “minimally invasive autopsy” provided equivalent information to autopsy
12 cases where “minimally invansive autopsy” provided inferior information than autopsy
2 cases where MRI and biopsy provided superior information
However, 73% of minimally invasive autopsies provided Information of equivalent clinical significance to autopsy
Fligner 2011
Alternatives to autopsyX ray
◦Useful for skeletal dysplasias◦Ribs/vertebrae as part of diagnosis of VACTERL or
diabetic fetopathy
X ray
Achondrogenesis 1b (absence of vertebral bodies)
Fligner 2011
X ray
Hemivertebrae (VACTERL)
Fligner 2011
EXAMINATION OF THE PLACENTA
Suggested workup of stillbirth
Examination of the placentaThe most valuable diagnostic test in most
studies◦Dutch study showed it to be valuable in 95% of
casesProvides additional information in 30% of
cases
Examination of the placentaWeight in relation to norms for gestational
ageEvidence of abruption, infarction,
thrombophiliasHemosiderin depositschronic abruption
◦Perivillious and marginal fibrin deposition◦Decidual necrosis◦Evidence of infarction
Examination of the placentaMultiples: chorionicity, vascular anastomoses
in multifetal gestationsCord: thrombosis, velamentous cord
insertion, vasa previaEvidence of infections
◦More common in preterm stillbirth◦Viral nuclic acid amplification◦Bacterial cultures
Examination of the placenta
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
GENETICSWorkup of stillbirth
KaryotypeAbnormal fetal karyotype noted in 8-13% of
all stillbirths and in >20% of those with morphologic abnormalities or IUGR
Dutch study: 11.9% prevalence of a chromosomal abnormality in the 362 IUFDs who underwent karyotyping◦37% trisomy 21, 16% monosomy X, 4% trisomy 13
Karyotype was valuable in 29% of cases
Cell cultureSuccessful in only ~50% of casesCells must be cultured in vitro—need
metaphase chromosomesOnly 1% of cells are dividing at any time
Kumar: Robbins pathologic basis of disease, 8th edition. 2009
Cell cultureAcceptable cytologic specimens:
◦Amniotic fluid (yield is 84%) obtained by amniocentesis at the time of prenatal diagnosis of demise, 20-30mL
◦1x1cm placental block from below the cord insertion site on an unfixed placenta
◦1.5cm segement of umbilical cord◦Internal fetal tissue specimen (costochondral
junction, patella, lung, kidney, gonad, not skin)
Cell cultureCollect the specimen in a sterile manner
◦Prokaryotic cells outcompete and overgrow any human cells
Transport to the laboratory as soon as possible
Blood, amniotic fluid, chorionic villi at room temperature
Solid tissue transported on wet ice◦Lysosomal enzyme activity is inhibited at 4 C and
viability is maintained
Cell cultureMonolayer in situUp to 2 weeks for solid tissue cultureCells are swelled hypotonically, ruptured
(blowing on the coverslip) and metaphase chromosomes are spread
Automatic robotic harvestersStained with Giemsa or Wright
◦Results in banding pattern unique to each chromosome
KaryotypeCount the chromosomes in
each cell◦ Technologist at a microscope
Must analyze 15-20 cells to get karyotype
Photograph of karyogram or computer-assisted imaging◦ Manipulate/move
chromosomes◦ Pattern-recognition software to
generate karyogram◦ Computer must be double-
checked by a technologistStein 2011
If culture is unsuccessfulFlourescent in-situ hybridization (FISH)
◦Can test for the most common aneuploidies◦Can test for specific mutations
FISHMolecular probe
◦Fluorescent dye ◦Visualized using fluorescence microscopy
Molecular probes can be◦Specific to certain mutations◦A cocktail that coats the whole chromosome
FISH can be performed on metaphase or interphase chromosomes—cell culture not required for FISH
FISHDNA is denatured and allowed to hybridize
with the probeNonhybridized DNA is then counterstained
FISH
X, X
Trisomy 21
Normal chromosome 21
Stein 2011
FISH
Microdeletion in chromosome 22 associated with DiGeorge syndrome
Stein 2011
FISH
Probes must be known genes/mutations
Limited number of colors can be seen with fluorescent microscope
Does not require metaphase chromosomes
Can obtain karyotype and information about mutations
Advantages Disadvantages
FISH with computer-assisted system
Trisomy 18 and XXY
•Fluorochromes can be mixed•Too subtle for human eye, but computer assisted systems allow detection of all 24 chromosomes•Karyotype in interphase
Stein 2011
If live cells are not available: MicroarrayScreens the genome for copy number
variations (CNPs)◦BAC arrays provide overview of genome◦SNP arrays provide more detailed coverage with
probes on every 100-1000 base pairs◦Detects deletions, duplications, aneuploidies, unbalanced translocations with a gain/loss of sequences
Good for small deletions or cryptic changes◦Cytogenetics resolution is only 5-10Mb
Microarray
Stein 2011
LimitationsMany copy number changes are of uncertain
clinical significance◦More CNVs are being identified every day
It will not detect balanced translocations or inversions
Microarray versus karyotypingReddy UM et al, 2012: Prospective
population-based study of 532 stillbirths over 2 years
Patients with IUFD underwent:◦Interview, chart abstraction, postpartum
examination, placental pathology, karyotype analysis, and specimen collection
DNA analyzed with an SNP microarray with data aligned to Human Genome release 18
Microarray versus karyotypingBenign CNV: full length listed in any of three
databases of unaffected personsPathogenic CNV:
◦Evidence of pathogenicity in literature◦A gene listed in the OMIM database that is known to
cause developmental/lethal disease◦In a pathogenic database
Probably benign CNV: no evidence of pathogenicity in literature
Unknown CNV
Microarray versus karyotypingMicroarray analysis yielded a result in 87.4%
stillbirths compared to 70.5% for karyotype85.2% of these were benign, too small, or
probably benign2.6% were pathogenic, 6.9% were aneuploidMicroarray detected CNV consistent with
DiGeorge syndrome not detected by karyotype in 3 cases
Conclusions: microarray
Can detect mutations too small for FISH
Higher chance of obtaining a result
More expensiveAdvantages DisadvantagesGenetic workup should be guided by family history and presence of congenital anomalies
Consider microarray when karyotype fails to grow in culture
MATERNAL WORKUPWorkup of stillbirth
IMMEDIATE MATERNAL WORKUPWorkup of stillbirth
Laboratories (Recommended)CBCKliehauer-BetkeHuman parvovirus B-19 IgG and IgMLupus anticoagulant, anticardiolipin
antibodiesIndirect Coombs
◦If not already done antepartumToxicology screen
Kliehauer BetkeRecommended to do before induction of
laborHowever, given that only massive
hemorrhage is likely to cause fetal death, can also be done up to 2-3 weeks after delivery
In Dutch study, FMH was a contributing factor in 10.6% of the total cohort
Kliehauer Betke
Reddy et al 2009
Antiphospholipid antibodiesOne fetal death satisfies criteria for testingConfirm with repeat testing in 6-12 weeksMore likely positive if stillbirth was accompanied
by IUGR or severe preeclampsiaTwo dutch studies (750 fetal deaths in Korteweg et
al 2010, 1025 fetal deaths in Korteweg et al 2012) showed that neither testing for acquired nor inherited thrombophilia is valuable◦Unless the patient has a family or personal history of
thrombophilia
Given significant implications for future pregnancies and future health of the mother, testing is generally recommended
Laboratories (Sometimes useful)SyphilisTSHInherited thrombophilia workup
◦Factor V Leiden, prothrombin gene mutation, antithrombin III, fasting homocysteine
Glucose screeningSonohysterogram
◦Especially if loss associated with preterm labor, PPROM, cervical insufficiency, previable gestations, fetal malpresentation
Guided by maternal history and risk factors
Inherited thrombophiliaKorteweg et al 2010. Multicenter, prospective study.
750 singleton fetal deaths >=20 wks, excluding terminations
Tested for vWF, antithrombin, protein C, total and free protein S, prothrombin gene mutation, factor V Leiden
Cause of death classified by a panel“Except for vWF and paternal free protein S,
acquired and thrombophilic defects were not more prevalent after fetal death.”
However, many case-control studies show an association
Consider it in cases of severe placental pathology, IUGR, or a history of thrombosisHowever, remember that most women with these conditions will have uncomplicated pregnancies
Laboratories (unproven benefit)Toxoplasmosis, rubella, CMV, HSV, other
infections◦Viruses for which vaccines are prevalent are
uncommon in developed countries◦However, if autopsy, pathology, or history is suggestive, take maternal/neonatal serology, special tissue staines, testing for nucleic acids
ANA
Routine TORCHDutch study: of the women tested for TORCH,
17.0% had positive IgM for TORCH, parvo, syphilis, or hepatitis B
However only 1.8% of the total cohort had infection as the cause of death
POSTPARTUM MATERNAL WORKUP
Suggested workup of stillbirth
In selected casesPaternal karyotypeProtein S and C activityKliehauer-Betke
ConsiderationsEvaluation of stillbirth
AIMMC protocolAll intact fetal remains are sent to the
morgue within 4 hours of expiration, UNLESS a Surgical Pathology examination is requested
If a Surgical Pathology examination is requested◦Remains with a Surgical Pathology requisition to
the Histology Laboratory◦Central Transport and Nursing are responsible for
the initial transport of the body to the morgue and filling out the morgue census sheet
AIMMC protocolIf autopsy is requested, the fetus is obtained
from the morgueAn autopsy is an option for fetuses >=20
weeks◦But if clinically indicated or requested by the
family, an autopsy can be offered before 20 weeks
AIMMC protocolAutopsies are performed by AIMMC
pathologists◦Trained in fetal/neonatal autopsies◦Consult specialists when needed
Preliminary result in 24 hours; final result in 30 days
Genetic and molecular testing at ACLA fetus that undergoes an autopsy can still
receive private burial
Fetal death disposition notificationWithin 24 hours after an
abortion/miscarriage the mother must be informed of her right to determine the final disposition of the remains◦Private burial◦Cremation◦Hospital disposition
Fetal death disposition notification
http://www.idph.state.il.us/vitalrecords/pdf/Fetal%20Death%20Disposition%20Notification.pdf
Fetal death disposition notificationIf patient is unclear, sign the form without
making a selection◦RN notes “undecided at discharge”
Fax a copy to Mission and Spiritual Care at 773-296-5010 and place a copy in the chart
Mission and Spiritual Care or the Perinatal Loss Coordinator will follow up with the family
If the family can not be reached within 2 weeks, Pathology will initiate the procedure for hospital disposition
Private burialPerinatal loss coordinator has a list of funeral homesCremation or burialOnly a licensed funeral director can pick up the
fetus◦Parents provide the name to Health Information
ManagementCost: some funeral home directors reduce the rate
◦Medicaid pays for modest funeral and burialFor very young fetuses, parents advised that
there might not be many remains after autopsy
Hospital dispositionSt Luke’s Cemetery
◦5300 N. Pulaski Road, Chicago, IL 60630“Babyland,” exact location not identifiedNo charge
Perinatal loss coordinatorAlways notifiy Anna Zieba (pager 61-7656,
phone 61-7127)Always notify Mission and Spiritual Care 61-
5005◦Can provide religious services from most religions
ConsiderationsParents benefit from seeing/holding the
infant◦Warn them about how the baby will appear
Use the term “baby”Encourage parents to name the infant
◦Knowing the sex is importantFetal loss can be devastation at any
gestational ageDifferent cultures grieve in different ways
ConsiderationsKeepsakes and memory boxesChild Life specialist can aid with siblingsFor a loss in the ER, consider consulting
crisis if parents show signs of depressionBereavement care track after discharge
ConclusionsEvaluation of stillbirth
ConclusionsThe cause of a stillbirth is the initial
pathophysiologic entity that irreversibly led to fetal death
Cause must be proven with evidence of fetal harm
There are many benefits to finding a causeEncourage patients to allow an evaluation
within the boundaries of their personal and cultural values
ConclusionsRecommended laboratories are CBC,
Kliehauer-Betke, parvovirus B-19 IgG and IgM, lupus anticoagulant, anticardiolipin antibodies, and toxicology screen
Only perform other labs as indicated by maternal history
Encourage patients to receive an autopsy◦Partial autopsy and MRI are alternatives
Always send the placenta to pathology
ConclusionsEncourage karyotypingBase evaluation for other chromosomal
abnormalities on family history and results of other studies
Consider FISH or microarray if low likelihood of getting a result from cell culture
Utilize hospital resources for perinatal loss
THANK YOU
SourcesEvaluation of stillbirth
ACOG Practice Bulletin Number 102. “Management of Stillbirth.” American College of Obstetricians and Gynecologists. March 2009. acog.org
Fligner CL, Dighe M. “Fetal and Perinatal Death Investigation: Redefining the Autopsy and the Role of Radiologic Imaging.” Ultrasound Clin 6, 2011 (105-117).
Illinois Masonic Medical Center Perinatal Loss Policy. Policy 20.118.044 Korteweg FJ, Erwich JJHM, Timmer A, et al. “Evaluation of 1025 fetal deaths: proposed diagnostic
workup.” Am J Obstet Gynecol 2012; 206:53.e1-12.Korteweg FJ, Erwich JJHM, et al. “Prevalence of Parental Thrombophilic Defects after Fetal Death and Relation to Cause.” Obstet and Gynecol. August 2010, Vol 116, No 2, part 1.
Kumar: Robbins and Coltran Pathologic Basis of Disease, Professional Edition, 8th ed. 2009 Saunders, an imprint of Elsevier.
Reddy UM, Goldenburg R, et al. “Stillbirth Classification—Developing an International Consensus for Research.” Obstet and Gynecol. Oct 2009, Vol 114, No 4.
Reddy UM, Page GP, Saade GR. “Karyotype versus Microarray testing for Genetic Abnormalities after Stillbirth.” N Engl J med 367; 23, December 2012
Silver R, Heuser C. “Stillbirth Workup and Delivery Management.” Clin Obstet and Gynecol 2010; 53, 3.
Stein, CK. “Applications of cytogenetics in Modern Pathology.” McPherson: Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd ed. 2011 Saunders, an imprint of Elsevier.