Antiphospholipid Antibodies in Stillbirth

Antiphospholipid Antibodies in Stillbirth

Robert M. Silver, MD, Corette B. Parker, PhD, Uma M. Reddy, MD, MPH, Robert Goldenberg, MD,Donald Coustan, MD, Donald J. Dudley, MD, George R. Saade, MD, Barbara Stoll, MD,Matthew A. Koch, MD, PhD, Deborah Conway, MD, Radek Bukowski, MD, Carol J. Rowland Hogue, PhD,Halit Pinar, MD, Janet Moore, MS, Marian Willinger, PhD, and D. Ware Branch, MD

OBJECTIVE: To compare antiphospholipid antibodies in

deliveries with and without stillbirth using a multicenter,

population-based case–control study of stillbirths and

live births.

METHODS: Maternal sera were assayed for immuno-

globulin (Ig)G and IgM anticardiolipin and anti-b2-glyco-

protein-I antibodies. Assays were performed in 582

stillbirth deliveries and 1,547 live birth deliveries.

RESULTS: Elevated levels of IgG anticardiolipin and IgG

anti-b2-glycoprotein-I antibodies were associated with

an approximate threefold increased odds of stillbirth

(crude odds ratio [OR] 3.43, 95% confidence interval

[CI] 1.79–6.60, 3.8% compared with 1.1% and OR 3.17,

95% CI 1.30–7.72, (1.9% compared with 0.6%, respec-

tively) when all deliveries with stillbirth were compared

with all deliveries with live birth. When the subset of

stillbirths not associated with fetal anomalies or obstetric

complications was compared with term live births, ele-

vated IgG anticardiolipin antibodies were associated with

stillbirth (5.0% compared with 1.0%; OR 5.30, 95% CI,

2.39–11.76; IgG anti-b2-glycoprotein-I antibodies (1.9%

compared with 0.6%) had an OR of 3.00 (95% CI 1.01–

8.90) and IgM anticardiolipin antibodies (6.0% compared

with 3.0%) had an OR of 2.03 (95% CI 1.09–3.76). Ele-

vated levels of anticardiolipin and anti-b2-glycoprotein-I

antibodies were associated with a threefold to fivefold

increased odds of stillbirth.

CONCLUSIONS: Our data support consideration of

testing for antiphospholipid antibodies in cases of other-

wise unexplained stillbirth.

(Obstet Gynecol 2013;122:641–57)

DOI: 10.1097/AOG.0b013e3182a1060e

LEVEL OF EVIDENCE: II

Antiphospholipid antibodies are a heterogeneousgroup of autoantibodies that have been associated

with thromboembolism and obstetric complicationsincluding stillbirth.1–3 Numerous antiphospholipid anti-bodies have been described but the best characterized,most widely recognized, and most strongly associatedwith clinical problems are the lupus anticoagulant, anti-cardiolipin antibodies and anti-b2-glycoprotein-I anti-bodies.1,4 Obstetric complications associated withantiphospholipid antibodies are thought to be attribut-able in part to placental insufficiency either from abnor-mal placental development or placental damage from

For a list of centers participating in the Stillbirth Collaborative ResearchNetwork, see the Appendix online at http://links.lww.com/AOG/A412.

From the University of Utah School of Medicine, Salt Lake City, Utah; RTIInternational, Research Triangle Park, North Carolina; the Pregnancy andPerinatology Branch, Eunice Kennedy Shriver National Institute of ChildHealth and Human Development, National Institutes of Health, Bethesda,Maryland; Columbia University, New York, New York; Brown UniversitySchool of Medicine, Providence, Rhode Island; the University of Texas HealthScience Center at San Antonio, San Antonio, Texas; the University of TexasMedical Branch at Galveston, Galveston, Texas; Emory University School ofMedicine, Atlanta, Georgia; and Rollins School of Public Health, Emory Uni-versity, Atlanta, Georgia.

Supported by grants from the Eunice Kennedy Shriver National Institute ofChild Health and Human Development: U10-HD045953 Brown University,Providence, Rhode Island; U10-HD045925 Emory University, Atlanta, Geor-gia; U10-HD045952 University of Texas Medical Branch at Galveston, Gal-veston, Texas; U10-HDO45955 University of Texas Health Sciences Center atSan Antonio, San Antonio, Texas; U10-HD045944 University of Utah HealthSciences Center, Salt Lake City, Utah; and U01-HD045954 RTI International,Research Triangle Park, North Carolina.

The authors thank the following members of the Eunice Kennedy ShriverNational Institute of Child Health and Human Development Scientific Advisoryand Safety Monitoring Board for their review of the study protocol, materials,and progress: Reverend Phillip Cato, PhD; James W. Collins, Jr, MD, MPH;Terry Dwyer, MD, MPH; William P. Fifer, PhD; John Ilekis, PhD; MarcIncerpi, MD; George Macones, MD, MSCE; Richard M. Pauli, MD, PhD;Raymond W. Redline, MD; Elizabeth Thom, PhD (chair) as well as all of theother physicians, study coordinators, research nurses, and patients who partici-pated in the Stillbirth Collaborative Research Network. The authors also thankDr. Huixia Yu for assistance in performing the assays and Susan Fox forassistance in the preparation of this manuscript.

Corresponding author: Robert M. Silver, MD, Department of Obstetrics andGynecology, University of Utah School of Medicine, 30 North 1900 East, Room2B308, Salt Lake City, UT 84132; e-mail: [email protected].

Financial DisclosureThe authors did not report any potential conflicts of interest.

© 2013 by The American College of Obstetricians and Gynecologists. Publishedby Lippincott Williams & Wilkins.ISSN: 0029-7844/13

VOL. 122, NO. 3, SEPTEMBER 2013 OBSTETRICS & GYNECOLOGY 641

inflammation, thrombosis, and infarction.5 Patients withclinical problems such as thrombosis (arterial, venous,or small vessel) and obstetric complications (unex-plained fetal death, three or more unexplained earlypregnancy losses, or severe placental insufficiency) aswell as specified levels of antiphospholipid antibodies(lupus anticoagulant, greater than 99% of anticardioli-pin, greater than 99% of anti-b2-glycoprotein-I) are con-sidered to have antiphospholipid antibody syndrome.2

Most studies of antiphospholipid antibody syn-drome and pregnancy loss have focused on recurrentearly pregnancy loss because the vast majority ofpregnancy losses occur during the pre-embryonic andembryonic periods.6–8 Nonetheless, many expertsconsider antiphospholipid antibodies to be morestrongly associated with fetal loss occurring duringthe fetal period (after 10 weeks of gestation).9 Many,if not most, studies of antiphospholipid antibodies donot provide details regarding the gestational age ofpregnancy loss. Some studies have included somefetal losses, typically during the second trimester.4,9,10

However, the association between antiphospholipidantibodies and stillbirth has not been systematicallyassessed.11 Thus, our objective was to compare mater-nal levels of antiphospholipid antibodies in womenwith and without stillbirth in a large, population-based, geographically and ethnically diverse cohort.

MATERIALS AND METHODS

A population-based case–control study of stillbirth wasconducted by the Stillbirth Collaborative ResearchNetwork of the Eunice Kennedy Shriver National Instituteof Child Health and Human Development. Womenwere enrolled at the time of delivery between March2006 and September 2008. The study was approved bythe institutional review boards of each clinical site andthe data coordinating center, and all participants gavewritten informed consent. Details of methods and studydesign have previously been published.12

Stillbirth was defined as Apgar scores of 0 at1 minute and 5 minutes and no signs of life by directobservation at 20 or more weeks of gestation.However, fetal deaths at 18 weeks or 19 weekswithout good dating also were included in the studyso as to not miss any potential cases at or beyond 20weeks of gestation.12 Gestational age was determinedby the best clinical estimate using multiple sourcesincluding assisted reproductive technology with doc-umentation of the day of ovulation of embryo transfer(if available), first day of the last menstrual period, andresults of obstetric ultrasonography.13 Deliveriesresulting from the termination of a live fetus wereexcluded.

The sample size considerations for the studyhave been described previously.14 Attempts weremade to enroll all deliveries with stillbirth (cases)and a contemporaneous representative sample ofdeliveries with live birth (controls) to women resid-ing in Stillbirth Collaborative Research Networkcatchment areas during the enrollment period. Thecatchment areas included 59 tertiary care and com-munity hospitals in portions of five states: RhodeIsland, Massachusetts, Georgia, Texas, and Utah.The areas were defined by state and county bound-aries, and the 59 hospitals within the catchment areasdeliver a combined total of more than 80,000 neo-nates per year. Some subgroups of live births were“oversampled” to ensure adequate numbers for strat-ified analyses.12

All women in the case group and women in thecontrol group had a standardized maternal interviewduring the delivery hospitalization and detailed chartabstraction of prenatal office visits, antepartum hos-pitalizations, and the delivery hospitalization. Mater-nal race was self-reported. Women in the case groupand women in the control group also had a uniformplacental pathology evaluation, and women in thecase group had a comprehensive standardized fetalpostmortem examination.15,16 Both were performedby a perinatal pathologist. Treating physicians wereadvised to obtain clinically recommended tests17

including anticardiolipin antibodies and lupus antico-agulant in cases of stillbirth. However, such testingwas performed at the discretion of the clinicians andwas not done in each case. In addition, attempts weremade to collect maternal blood for serum and DNA,fetal blood from the umbilical cord (when available),placental tissue in women in the case group andwomen in the control group, and fetal tissue in womenin the case group around the time of delivery orenrollment. Maternal serum was stored at 280°C for2–5 years before assay.

Maternal serum samples in all women in the casegroup (regardless of whether or not they had clinicallyindicated antiphospholipid antibody testing) and allwomen in the control group (with adequate sample)were tested for antiphospholipid antibodies in theBranch Perinatal Laboratory (University of Utah HealthSciences Center, Salt Lake City, Utah). Serum sampleswere tested for anticardiolipin and anti-b2-glycoprotein-Iimmunoglobulin (Ig)G and IgM antibodies using kitsfrom INOVA Diagnostics. Testing was performed induplicate by experienced laboratory personnel blindedto patients’ clinical diagnosis using procedures recom-mended by the manufacturer. The thresholds todefine elevated values for the anti-b2-glycoprotein-I and

642 Silver et al Antiphospholipid Antibodies in Stillbirth OBSTETRICS & GYNECOLOGY

anticardiolipin assays were determined by the manufac-turer using a percentile-based method (99% or more ofa healthy population) as previously described.18,19 Posi-tive results were defined as 20 or more units IgG anti-cardiolipin antibodies, 20 or more units IgManticardiolipin antibodies, 20 or more units IgG anti-b2-glycoprotein-I antibodies, and 20 or more units IgManti-b2-glycoprotein-I antibodies.

The Initial Causes of Fetal Death system devel-oped by the Stillbirth Collaborative Research Net-work was used to assign causes of death to each case.20

Each case of stillbirth was reviewed by two physicians,and difficult cases were evaluated and adjudicated bya multidisciplinary panel with expertise in geneticsand perinatal pathology.21 Some analyses were per-formed on subsets of cases and controls, includingnonanomalous stillbirths and stillbirths withoutobstetric complications as previously described.14

Nonanomalous stillbirths excluded those stillbirthswith possible or probable causes of stillbirth thatincluded fetal genetic, structural, and karyotypicabnormalities.20 Cases with possible or probablecauses of stillbirth including fetal–maternal hemor-rhage, cervical insufficiency, preterm labor, pretermpremature rupture of membranes, clinical chorioam-nionitis, intrapartum death, abruption, complicationsof multiple gestation, and uterine rupture wereexcluded in the subset of stillbirths without obstetriccomplications.20

The delivery, defined as a case if there were anystillbirths delivered and as a control if all live birthswere delivered, was the unit of analysis. The analyseswere weighted for oversampling and other aspects ofthe study design as well as for differential consent usingSUDAAN 10.0 software.22 Construction of the weightshas been previously described.12 Crude and adjustedodds ratios (ORs) and 95% confidence intervals (CIs)were calculated from univariate and multivariablelogistic regression models. All tests were performedat a nominal significance level of a50.05. All singledegree-of-freedom tests were two-sided.

The adjusted ORs account for stillbirth riskfactors known at pregnancy confirmation (baseline)using a modification to a risk factor score for stillbirththat was developed on the logit scale using thecoefficients from a logistic regression model. Themodel used data on all Stillbirth CollaborativeResearch Network deliveries where a maternal inter-view was conducted and a prenatal chart wasabstracted. Variables contributing to the baselinerisk factor score were those described previously14

and included the following maternal characteristics:age, race and ethnicity, marital status, education,

pregnancy history, body mass index, smoking status,alcohol use, illicit drug use, hypertension, diabetes,seizure disorder, blood type, Rh factor, and multiplegestation in current pregnancy as well as paternal age,

Eligible stillbirth deliveriesN=953

Enrolledn=663; 70%

Maternal serum test forantiphospholipid antibodies

n=582; 88%

Stillbirth without anomaliesn=390

Stillbirth without anomaliesor obstetric complications

n=273

Excluded: n=290Not approached: 126Declined: 164

Did not consent to blooddraw or serum unavailable

n=81

A

Eligible live birth deliveriesN=3,088

Enrolled n=1,932; 63%

Maternal serum test forantiphospholipid antibodies

n=1,547; 80%

Term live births n=1,164

Excluded: n=1,156Not approached: 394Declined: 762

Did not consent to blooddraw or serum unavailable

n=385

B

spuorgbus sisylanA

spuo rgbus sisylanA

Fig. 1. Study enrollment and inclusion in antiphospholipidantibody analysis for (A) stillbirth deliveries and (B) livebirth deliveries. A pregnancy was categorized as a stillbirthpregnancy if there were any stillbirths delivered and asa live birth pregnancy if all live births were delivered. Afetal death was defined by Apgar scores of 0 at 1 and5 minutes and no signs of life by direct observation. Fetaldeaths were classified as stillbirths if the best clinical esti-mate of gestational age at death was 20 weeks of gestationor more. Fetal deaths at 18 and 19 weeks of gestationwithout good dating were also included as stillbirths. Theanalysis includes comparison of all stillbirths to all livebirths, all stillbirths to term live births, nonanomalousstillbirths to term live births, and nonanomalous stillbirthswithout obstetric complications to term live births.

Silver. Antiphospholipid Antibodies in Stillbirth. Obstet Gynecol2013.

VOL. 122, NO. 3, SEPTEMBER 2013 Silver et al Antiphospholipid Antibodies in Stillbirth 643

family income, insurance and method of payment,and clinical site. All variables included in the scorewere categorical and a weighted average of the regres-sion coefficients associated with the categories wasused when a variable was missing for an observation.The weights were taken as the sample weighted pro-portion of live births by category. There were veryfew missing values, as previously noted.14 The mod-ification to the risk factor score for this analysis was

to exclude coefficients associated with pregnancyhistory.

RESULTS

Figure 1 depicts enrollment to the Stillbirth Collabo-rative Research Network and inclusion in this analy-sis. A total of 582 deliveries with stillbirth and 1,547with live births had blood available and assessment ofanticardiolipin and anti-b2-glycoprotein-I antibody

Table 1. Comparison of Women Who Were Tested for Antiphospholipid Antibodies in Serum and ThoseWho Were Not Tested by Pregnancy Outcome

Characteristic

Stillbirths Live Births

Antibody Testing

P

Antibody Testing

PYes No Yes No

Unweighted sample size (n) 582 81 1,547 385Weighted sample size (n) 579 84 1,184 255Maternal age at delivery (y)

Younger than 20 13.0 14.5 .731 9.5 13.9 .02420–34 70.1 66.9 76.5 71.735–39 12.0 15.4 12.3 10.240 and older 4.9 3.3 1.7 4.1

Maternal race and ethnicityWhite, non-Hispanic 35.7 17.7 ,.001 47.4 38.6 ,.001Black, non-Hispanic 20.7 41.6 9.4 22.6Hispanic 37.0 31.5 36.0 29.4Other 6.6 9.1 7.2 9.4

Marital statusNot married or cohabitating 25.0 27.7 .648 14.9 17.3 .454Cohabitating 25.5 29.1 23.7 25.2Married 49.5 43.2 61.4 57.5

Maternal education (y)0–11 (none, primary, some secondary) 23.5 25.8 .669 18.3 18.5 .81612 (completed secondary) 29.2 33.0 25.5 27.413 or more (college) 47.3 41.2 56.2 54.1

Insurance and method of paymentNo insurance 5.3 10.1 .238 3.6 3.2 .439Any public or private assistance 53.8 51.8 47.9 52.3Veterans’ Administration, commercial health

insurance, or health maintenance organization40.9 38.2 48.5 44.6

IncomeOnly public or private assistance 9.3 3.7 .356 5.9 5.9 .704Assistance and personal income 38.0 36.8 37.0 39.8Only personal income 52.7 59.5 57.1 54.2No 1st- or 2nd-trimester prenatal care 7.3 12.7 .120 2.5 4.7 .031

Gestational age (wk)18–19 2.3 3.8 .137 0.0 0.0 .25320–23 32.0 46.8 0.4 0.324–27 15.9 15.6 0.7 1.028–31 13.1 10.3 1.0 1.232–26 19.8 11.7 8.2 10.637 or more 16.9 11.9 89.8 86.9Nulliparous 43.3 56.7 .029 34.6 38.9 .213Multifetal pregnancy 6.5 4.3 .421 1.7 2.9 .240

Data are % unless otherwise specified.Results are weighted for the study design and differential consent based on characteristics recorded in the screened population. Overall

sample sizes are given, unweighted and weighted. Sample sizes for the weighted percentages vary slightly by characteristic.


levels. Women in the case group who did and did notenroll in the Stillbirth Collaborative Research Networkwere similar with regard to maternal age, maternal raceand ethnicity, insurance and method of payment, andgestational age at delivery. Women in the control groupwho did not enroll differed from those who enrolled bymaternal race and ethnicity and gestational age at deliv-ery.21 The demographic characteristics of the StillbirthCollaborative Research Network study participantshave been published.14,21 Among women with stillbirth,those women refusing blood draw were more likely tobe non-Hispanic black and nulliparous than those con-senting (Table 1). Women with live births who werenon-Hispanic black, younger than 20 years or 40 yearsand older, and did not receive early prenatal care weremore likely to refuse blood draw (Table 1).

Abnormal levels of anticardiolipin and anti-b2-glycoprotein-I antibodies in all stillbirths compared withall live births are shown in Table 2. The proportion ofdeliveries with elevated levels of IgG anticardiolipinantibodies was higher in women in the case group com-pared with women in the control group (3.8 comparedwith 1.1%, OR 3.43, 95% CI 1.79–6.60). The same wastrue for IgG anti-b2-glycoprotein-I antibodies (OR 3.17,95% CI 1.30–7.72). However, similar proportions of

deliveries with stillbirth and with live births had elevatedlevels of IgM anticardiolipin and anti-b2-glycoprotein-Iantibodies. The OR for at least one positive antibodytest was 1.63 (95% CI 1.13–2.35). Results were similarfor adjusted odds ratios (Table 2).

Table 2 also shows levels of anticardiolipin and anti-b2-glycoprotein-I antibodies in all stillbirths comparedwith term live births. Results were similar to the com-parison between all stillbirths and all live births. Womenhaving a stillbirth were more likely to have elevatedlevels of IgG anticardiolipin and anti-b2-glycoprotein-Iantibodies than women having a term live birth.

Similar results also were noted when the subset ofnonanomalous stillbirths was compared with term livebirths (Table 3). A greater percentage of women withnonanomalous stillbirths had elevated levels of IgGanticardiolipin antibodies (4.8%) compared with thosewith term live births (1.0%, OR 5.09, 95% CI 2.44–10.65). A positive test for IgG anti-b2-glycoprotein-Iantibodies yielded an OR of 2.87 (95% CI 1.05–7.88)for stillbirth. Positive tests for IgM anticardiolipin andIgM anti-b2-glycoprotein-I antibodies were similarbetween groups. Having one or more positive testfor an antibody was associated with a 2.02 OR (95%CI 1.34–3.05) for stillbirth.

Table 2. Abnormal Antiphospholipid Antibody Levels in All Stillbirths, All Live Births, and Term Live Births

Antibody StillbirthsLiveBirths

Term LiveBirths*

Stillbirths vs Live Births Stillbirths vs Term Live Births

Crude Adjusted† Crude Adjusted†

Unweighted samplesize (n)

582 1,547 1,164

Weighted samplesize (n)

579 1,184 1,063

IgG anticardiolipin 3.8 1.1 1.0 3.43 (1.79–6.60) 3.02 (1.47–6.21) 3.98 (1.96–8.07) 3.57 (1.64–7.74)IgM anticardiolipin 3.4 3.1 3.0 1.10 (0.63–1.92) 1.16 (0.63–2.11) 1.13 (0.64–2.00) 1.17 (0.62–2.21)IgG anti-b2-glycoprotein-I

1.9 0.6 0.6 3.17 (1.30–7.72) 3.04 (1.13–8.19) 3.03 (1.20–7.62) 2.91 (1.03–8.25)

IgM anti-b2-glycoprotein-I

2.7 1.9 1.9 1.42 (0.73–2.76) 1.07 (0.51–2.21) 1.45 (0.73–2.89) 1.06 (0.49–2.29)

Abnormal level of oneor more antibody

9.6 6.1 5.9 1.63 (1.13–2.35) 1.54 (1.04–2.29) 1.68 (1.15–2.45) 1.60 (1.06–2.43)

Ig, immunoglobulin.Data are % or odds ratio (95% confidence interval) unless otherwise specified.Abnormal antibody levels are defined as 20 or more units of IgG anticardiolipin antibodies, 20 or more units of IgM anticardiolipin

antibodies, 20 or more units of IgG anti-b2-glycoprotein-I antibodies, and 20 or more units of IgM anti-b2-glycoprotein-I antibodies.Results shown are weighted for the study design and differential consent based on characteristics recorded on all eligible pregnanciesthat were screened for the study. Overall sample sizes are given, unweighted and weighted.

* Defined as gestational age of 37 weeks of gestation or more at the time of birth.† The adjusted odds ratios account for prepregnancy risk factors for stillbirth using a modification to a score developed on the logit scale and

using data on all participants to the Stillbirth Collaborative Research Network. Variables contributing to the baseline risk factor scoreincluded the following maternal characteristics: age, race and ethnicity, marital status, education, pregnancy history, body mass index,smoking status, alcohol use, illicit drug use, hypertension, diabetes, seizure disorder, blood type, Rh factor, and multiple gestation incurrent pregnancy as well as paternal age, family income, insurance and method of payment, and clinical site. The modification to therisk factor score for this analysis is to exclude coefficients associated with pregnancy history. The score is available on 553 of the 582stillbirth pregnancies, 1,499 of the 1,547 live birth pregnancies, and 1,132 of the 1,164 term live birth pregnancies.


Table 3 also depicts antibody results in womenwith stillbirth not associated with fetal anomalies orobstetric complications compared with women withterm live births. IgG anticardiolipin antibodies wereassociated with a fivefold odds of stillbirth and IgManticardiolipin antibodies were associated with a two-fold odds of stillbirth. IgG anti-b2-glycoprotein-I anti-bodies were associated with a threefold odds ofstillbirth, but IgM anti-b2-glycoprotein-I antibodieswere not associated with stillbirth. Of these specificstillbirth deliveries, 11.7% had at least one positivetest for antiphospholipid antibodies compared with5.9% of those with term live births (OR 2.11, 95%CI 1.33–3.33).

We explored whether a threshold of 40 ormore units of IgG or IgM anticardiolipin or anti-b2-glycoprotein-I antibodies was more strongly associatedwith stillbirth than using a threshold of 20 or moreunits. For each of the four antibodies and in each ofthe subgroups of stillbirth and live birth analyzed,a threshold of 40 units was not more strongly associ-ated with stillbirth than a threshold of 20 units (datanot shown).

We then assessed the clinical characteristics ofthe cases with positive tests for antiphospholipidantibodies in hopes of identifying specific scenariosthat merit testing. Details for each case are shown inTable 4. In this study, there were 56 deliveries withstillbirth (including three sets of twins, one with two

stillbirths, and two with a live birth and a stillbirth)and at least one positive test for antiphospholipid anti-bodies. In these cases, a history of thrombosis wasnoted in one (1.8%) and none had systemic lupuserythematosus. Pregnancy complications included pre-eclampsia in 6 of 53 (11.3%), small-for-gestational-agefetus in 20 of 54 (37.0%), and clinical or histologicevidence of abruption in 14 of 56 (25.0%). Of the 50stillbirths with a complete postmortem examination,seven (14%) had antiphospholipid antibody syndromeas a probable cause of death based on Initial Causes ofFetal Death criteria. It is noteworthy that many othercases in this subset of 57 stillbirths had a likely cause ofdeath other than antiphospholipid antibody syndrome.One case of interest was congenital syphilis, which isknown to cause antiphospholipid antibody production.In this cohort of 56 women with positive tests for anti-phospholipid, 22 (39.3%) had no history of (prior) preg-nancy loss, thrombosis, systemic lupus erythematosus,small-for-gestational-age fetus, or preeclampsia. Thus,we were unable to ascertain clinical features that wouldallow testing only a subset of patients with stillbirth forantiphospholipid.

Another important question is whether it is neces-sary to test for all four antibodies assessed in this study.Forty-six of the 56 (82.1%) women having a positive testresult tested positive for only one of the four antibodies.Thus, not testing for any one of the antibodies wouldhave potentially missed some cases. Only 19 patients

Table 3. Abnormal Antiphospholipid Antibody Levels in Nonanomalous Stillbirths, NonanomalousStillbirths Without Obstetric Complications, and Term Live Births

Antibody Nonanomalous StillbirthsNonanomalous StillbirthsWithout Complications Term Live Births*

Unweighted sample size (n) 390 273 1,164Weighted sample size (n) 387 268 1,063IgG anticardiolipin 4.8 5.0 1.0IgM anticardiolipin 5.0 6.0 3.0IgG anti-b2-glycoprotein-I 1.8 1.9 0.6IgM anti-b2-glycoprotein-I 3.1 2.5 1.9Abnormal level of one or more antibody 11.3 11.7 5.9

Ig, immunoglobulin.Data are % or odds ratio (95% confidence interval) unless otherwise specified.Abnormal levels are defined as 20 or more units of IgG anticardiolipin antibodies, 20 or more units of IgM anticardiolipin antibodies, 20 or

more units of IgG anti-b2-glycoprotein-I antibodies, and 20 or more units of IgM anti-b2-glycoprotein-I antibodies. Results shown areweighted for the study design and differential consent based on characteristics recorded on all eligible pregnancies that were screenedfor the study. Overall sample sizes are given, unweighted and weighted.

* Defined as gestational age of 37 weeks of gestation or more at time of birth.† The adjusted odds ratios account for prepregnancy risk factors for stillbirth using a modification to a score developed on the logit scale and

using data on all participants to the Stillbirth Collaborative Research Network. Variables contributing to the baseline risk factor scoreincluded the following maternal characteristics: age, race and ethnicity, marital status, education, pregnancy history, body mass index,smoking status, alcohol use, illicit drug use, hypertension, diabetes, seizure disorder, blood type, Rh factor, and multiple gestation incurrent pregnancy as well as paternal age, family income, insurance and method of payment, and clinical site. The modification to therisk factor score for this analysis is to exclude coefficients associated with pregnancy history. The score is available on 377 of the 390nonanomalous stillbirth pregnancies, 262 of the 273 nonanomalous stillbirth pregnancies without obstetric complications, and 1,132 ofthe 1,164 term live birth pregnancies.


with stillbirth had clinically indicated testing for lupusanticoagulant, and two of these were positive.

DISCUSSION

Elevated levels of IgG anticardiolipin and anti-b2-

glycoprotein-I antibodies were associated with anapproximate threefold increased odds of stillbirthwhen all stillbirth deliveries are compared with all livebirth deliveries. However, levels of IgM anticardioli-pin and anti-b2-glycoprotein-I antibodies were similaramong groups. Women with stillbirth had an approx-imately 1.6-fold risk for having at least one positiveantibody compared with those with live births.

When the subset of stillbirths not associated withfetal anomalies or obstetric complications was com-pared with term live births, elevated IgG anticardio-lipin antibodies were associated with an OR of 5.30(95% CI 2.39–11.76) for stillbirth. The OR for IgManticardiolipin antibodies was 2.03 (95% CI 1.09–3.76) and the OR for IgG anti-b2-glycoprotein-I anti-bodies was 3.00 (95% CI 1.01–8.90). Stillbirth was notsignificantly associated with IgM anti-b2-glycopro-tein-I antibodies. The OR for stillbirth among womenwith one or more positive antibody test comparedwith none was 2.11 (95% CI 1.33–3.33).

These data confirm and quantify the previouslysuspected association between antiphospholipid anti-bodies and stillbirth. A meta-analysis of 25 studies notedan OR for late recurrent fetal loss of 3.57 (95% CI 2.26–5.65) for any positive test for anticardiolipin antibod-ies.23 The OR for recurrent pregnancy loss in womenwith medium-to-high titer IgG anticardiolipin antibod-ies was 4.68 (95% CI 2.96–7.40).23 Although somewomen with stillbirth were included in these studies,most pregnancy losses were before 20 weeks of gesta-tion, and all were less than 24 weeks of gestation.23

In addition, the women in the meta-analysis hadrecurrent rather than sporadic pregnancy loss. Incontrast, our study is one of the few to addresssporadic stillbirth. It is noteworthy that antiphospholipid

antibodies are associated with recurrent early pregnancyloss23 but not sporadic early pregnancy loss.24 This isexpected because sporadic early pregnancy loss is com-mon and usually the result of genetic abnormalities. Incontrast to early pregnancy loss, even a single fetal deathafter 20 weeks of gestation is considered to be clinicalevidence of antiphospholipid antibody syndrome. Thus,our data support the Sapporo obstetric criteria for anti-phospholipid antibody syndrome that include sporadicfetal death.

It is noteworthy that 56 of 582 (9.6%) women withstillbirth had positive tests for antiphospholipid anti-bodies. It is unclear that all of these women in the casegroup will prove to have antiphospholipid antibodysyndrome because antibody levels in many cases werelower than those considered diagnostic of antiphos-pholipid antibody syndrome and they were notsystematically assessed 12 weeks after the initial assay.Some cases had other potential causes of stillbirth, andmany had only modestly elevated levels of antibodies.There were many cases in which the only clinicalevidence of antiphospholipid antibody syndrome wasunexplained stillbirth. Fourteen of these women in thecase group had very high levels (greater than 40 units)of antibodies. Thus, it seems as though testing allwomen with unexplained stillbirth is a reasonableapproach to the workup of stillbirth. Also, althoughIgG anticardiolipin antibodies were most stronglyassociated with stillbirth, testing for all four antibodiesassessed in this study can identify additional womenwith potential antiphospholipid antibody syndromecompared with testing for IgG anticardiolipin anti-bodies alone.

It is important for clinicians to be aware that 6%of live births had at least one positive test forantiphospholipid antibodies. Thus, a positive test isnot diagnostic of antiphospholipid antibody syn-drome. It is critical to be sure that the stillbirth isotherwise unexplained when considering a diagnosisof antiphospholipid antibody syndrome. It is also

Nonanomalous Stillbirths ComparedWith Term Live Births

Nonanomalous Stillbirths Without ComplicationsCompared With Term Live Birth

Crude Adjusted† Crude Adjusted†

5.09 (2.44–10.65) 4.16 (1.84–9.40) 5.30 (2.39–11.76) 3.96 (1.74–8.98)1.66 (0.93–2.97) 1.68 (0.88–3.21) 2.03 (1.09–3.76) 1.99 (1.01–3.94)2.87 (1.05–7.88) 2.88 (0.99–8.35) 3.00 (1.01–8.90) 2.84 (0.93–8.66)1.68 (0.79–3.56) 1.31 (0.59–2.89) 1.32 (0.54–3.25) 1.12 (0.43–2.92)2.02 (1.34–3.05) 1.93 (1.24–2.99) 2.11 (1.33–3.33) 1.95 (1.21–3.15)


Table 4. Stillbirths With Positive Antiphospholipid Antibody Results

CaseNo.

PregnancyOutcomes

IgGAnticardiolipinAntibodies(Units)

IgMAnticardiolipinAntibodies(Units)

IgG Anti-b2-Glycoprotein-IAntibodies(Units)

IgM Anti-b2-Glycoprotein-IAntibodies(Units)

GestationalAge

Reported atScreening

(wk) SGA

1 Live birth/stillbirth 23 8 3 3 36 Yes/yes

2 Stillbirth/stillbirth 8 6 24 9 22 No/no

3 Stillbirth 10 7 21 5 26 Yes

4 Stillbirth 12 26 4 5 38 No










SGA, small for gestational age.These cases include 56 pregnancies with 57 stillbirths. Among these were three sets of twins.


PregnancyHistory

Thrombosis(ChartReview)

Preeclampsiaor GestationalHypertension

(ChartReview)

PlacentalAbruption(ChartReview)

RetroplacentalHematoma(PlacentaPathology)

LupusAnticoagulant

(ChartReview)

Cause of Death:Possible or ProbableAntiphospholipid

Antibody Syndrome(Initial Causes of

Fetal Death)

Nulliparous; neverpregnant or onlyelectiveterminations

No Yes No Not done Not done No

Multiparous with noprevious losses atless than 20 wk ofgestation orstillbirths

No No No Not done Not done No/no

Nulliparous withprevious losses

No Yes No No No No


No No No No Not done No


No No No Not done Not done Not done




No No No Yes Not done No


Yes No No No Not done Probable cause


No No Yes Yes Not done No


No No No No Not done Possible cause


No Yes No No Not done No


No No No No Not done Not done


No No No No No No

(continued )



CaseNo.

PregnancyOutcomes





GestationalAge


(wk) SGA






19 Stillbirth 7 30 61 12 21 Do not know





24 Stillbirth 66 19 29 24 20 No






(continued )


PregnancyHistory



(ChartReview)



LupusAnticoagulant

(ChartReview)



Fetal Death)

Multiparous with nostillbirth butprevious losses atless than 20 wk ofgestation

No No No No No No






No No Yes No Not done No


No No No Yes Not done Possible cause


No No No No No No


No No Yes Yes Not done No




No No No Not done Not done No


No No No Not done Not done Not done








No No No Yes Yes No



Muliparous withstillbirth

No Yes No Do not know No No

(continued )



CaseNo.

PregnancyOutcomes





GestationalAge


(wk) SGA

30 Stillbirth 151 56 111 5 27 No









39 Stillbirth 23 15 12 54 23 No





(continued )


PregnancyHistory



(ChartReview)



LupusAnticoagulant

(ChartReview)



Fetal Death)


No Yes No Yes Not done No














No No No Not done Not done No




No Do not know Yes Yes Not done No




No Yes Do not know No Not done No


No No No No Yes Probable cause


No No No No No No

(continued )



CaseNo.

PregnancyOutcomes





GestationalAge


(wk) SGA





48 Live birth/stillbirth 5 21 3 14 24 No/do not know









(continued )


PregnancyHistory



(ChartReview)



LupusAnticoagulant

(ChartReview)



Fetal Death)


No No No No No Not done


No Do not know Yes Yes No No


No No No Not done No No

Multiparous withstillbirth





No No No No No No


No No No No No No


No No No No No No


No No No No No No


No No No No No Possible cause

Multiparous withstillbirth

No Do not know Yes No No No






imperative to repeat testing because positive tests maybe transient. Finally, it should be understood that lowpositive test results (eg, levels between 20 units and 40units) are not diagnostic of antiphospholipid antibodysyndrome and that treatment is not proven to beefficacious in such cases.2

There were several weaknesses of our study. Wedid not assess lupus anticoagulant in each patientbecause we did not have available plasma. Second,women did not have serial assessment of antiphos-pholipid antibodies. These autoantibodies may fluc-tuate over time, and it is necessary to have elevatedlevels persist on two occasions at least 12 weeks apartto be considered diagnostic of antiphospholipid anti-body syndrome.2 Third, we did not have detailedinformation regarding the possible influence of treat-ment for antiphospholipid antibody syndrome as wemight be able to obtain in a longitudinal cohort study.Finally, the slight differences in women who did anddid not agree to blood draw may limit the generaliz-ability of our data.

The study also had numerous strengths. It is one ofthe few studies of antiphospholipid that was population-based, providing reliable estimates of the associationof stillbirth with antiphospholipid antibodies. It alsoinvolved a geographically, racially, and ethnicallydiverse population, enhancing the generalizability ofthe results. In addition, it is one of the largest studiesof antiphospholipid antibodies and stillbirth, includ-ing almost 600 women in the case group. Importantly,participants underwent an extensive evaluation forother potential causes of stillbirth. Finally, the labo-ratory performing the antiphospholipid antibodyassays was blinded to clinical status of the samples.

In summary, elevated levels of IgG anticardioli-pin and anti-b2-glycoprotein-I antibodies are associ-ated with a threefold to fivefold increased odds ofstillbirth. IgG anticardiolipin antibodies are morestrongly associated with stillbirth than IgM antibodies.Almost 10% of participants with stillbirth had positivetests for antiphospholipid antibodies, and several hadantiphospholipid antibody syndrome as a possible orprobable cause of death. Our data support consider-ation of testing for antiphospholipid in cases of other-wise unexplained stillbirth.

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References:

1. U.S. Departmentof Health and Human Services. National Institutes of Health publicaccess. Availableathttp://publicaccess.nih.gov/. Retrieved July 10, 2008.

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Antiphospholipid Antibodies in Stillbirth