Antiphospholipid antibodies are a heterogeneous group of autoantibodies that have been associated with thromboembolism and obstetric complications including stillbirth.1–3 Numerous antiphospholipid antibodies have been described but the best characterized, most widely recognized, and most strongly associated with clinical problems are the lupus anticoagulant, anticardiolipin antibodies and anti-β2-glycoprotein-I antibodies.1,4 Obstetric complications associated with antiphospholipid antibodies are thought to be attributable in part to placental insufficiency either from abnormal placental development or placental damage from inflammation, thrombosis, and infarction.5 Patients with clinical problems such as thrombosis (arterial, venous, or small vessel) and obstetric complications (unexplained fetal death, three or more unexplained early pregnancy losses, or severe placental insufficiency) as well as specified levels of antiphospholipid antibodies (lupus anticoagulant, greater than 99% of anticardiolipin, greater than 99% of anti-β2-glycoprotein-I) are considered to have antiphospholipid antibody syndrome.2
Most studies of antiphospholipid antibody syndrome and pregnancy loss have focused on recurrent early pregnancy loss because the vast majority of pregnancy losses occur during the pre-embryonic and embryonic periods.6–8 Nonetheless, many experts consider antiphospholipid antibodies to be more strongly associated with fetal loss occurring during the fetal period (after 10 weeks of gestation).9 Many, if not most, studies of antiphospholipid antibodies do not provide details regarding the gestational age of pregnancy loss. Some studies have included some fetal losses, typically during the second trimester.4,9,10 However, the association between antiphospholipid antibodies and stillbirth has not been systematically assessed.11 Thus, our objective was to compare maternal levels of antiphospholipid antibodies in women with and without stillbirth in a large, population-based, geographically and ethnically diverse cohort.
MATERIALS AND METHODS
A population-based case–control study of stillbirth was conducted by the Stillbirth Collaborative Research Network of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Women were enrolled at the time of delivery between March 2006 and September 2008. The study was approved by the institutional review boards of each clinical site and the data coordinating center, and all participants gave written informed consent. Details of methods and study design have previously been published.12
Stillbirth was defined as Apgar scores of 0 at 1 minute and 5 minutes and no signs of life by direct observation at 20 or more weeks of gestation. However, fetal deaths at 18 weeks or 19 weeks without good dating also were included in the study so as to not miss any potential cases at or beyond 20 weeks of gestation.12 Gestational age was determined by the best clinical estimate using multiple sources including assisted reproductive technology with documentation of the day of ovulation of embryo transfer (if available), first day of the last menstrual period, and results of obstetric ultrasonography.13 Deliveries resulting from the termination of a live fetus were excluded.
The sample size considerations for the study have been described previously.14 Attempts were made to enroll all deliveries with stillbirth (cases) and a contemporaneous representative sample of deliveries with live birth (controls) to women residing in Stillbirth Collaborative Research Network catchment areas during the enrollment period. The catchment areas included 59 tertiary care and community hospitals in portions of five states: Rhode Island, Massachusetts, Georgia, Texas, and Utah. The areas were defined by state and county boundaries, and the 59 hospitals within the catchment areas deliver a combined total of more than 80,000 neonates per year. Some subgroups of live births were “oversampled” to ensure adequate numbers for stratified analyses.12
All women in the case group and women in the control group had a standardized maternal interview during the delivery hospitalization and detailed chart abstraction of prenatal office visits, antepartum hospitalizations, and the delivery hospitalization. Maternal race was self-reported. Women in the case group and women in the control group also had a uniform placental pathology evaluation, and women in the case group had a comprehensive standardized fetal postmortem examination.15,16 Both were performed by a perinatal pathologist. Treating physicians were advised to obtain clinically recommended tests17 including anticardiolipin antibodies and lupus anticoagulant in cases of stillbirth. However, such testing was performed at the discretion of the clinicians and was not done in each case. In addition, attempts were made to collect maternal blood for serum and DNA, fetal blood from the umbilical cord (when available), placental tissue in women in the case group and women in the control group, and fetal tissue in women in the case group around the time of delivery or enrollment. Maternal serum was stored at −80°C for 2–5 years before assay.
Maternal serum samples in all women in the case group (regardless of whether or not they had clinically indicated antiphospholipid antibody testing) and all women in the control group (with adequate sample) were tested for antiphospholipid antibodies in the Branch Perinatal Laboratory (University of Utah Health Sciences Center, Salt Lake City, Utah). Serum samples were tested for anticardiolipin and anti-β2-glycoprotein-I immunoglobulin (Ig)G and IgM antibodies using kits from INOVA Diagnostics. Testing was performed in duplicate by experienced laboratory personnel blinded to patients' clinical diagnosis using procedures recommended by the manufacturer. The thresholds to define elevated values for the anti-β2-glycoprotein-I and anticardiolipin assays were determined by the manufacturer using a percentile-based method (99% or more of a healthy population) as previously described.18,19 Positive results were defined as 20 or more units IgG anticardiolipin antibodies, 20 or more units IgM anticardiolipin antibodies, 20 or more units IgG anti-β2-glycoprotein-I antibodies, and 20 or more units IgM anti-β2-glycoprotein-I antibodies.
The Initial Causes of Fetal Death system developed by the Stillbirth Collaborative Research Network 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 by a multidisciplinary panel with expertise in genetics and perinatal pathology.21 Some analyses were performed on subsets of cases and controls, including nonanomalous stillbirths and stillbirths without obstetric complications as previously described.14 Nonanomalous stillbirths excluded those stillbirths with possible or probable causes of stillbirth that included fetal genetic, structural, and karyotypic abnormalities.20 Cases with possible or probable causes of stillbirth including fetal–maternal hemorrhage, cervical insufficiency, preterm labor, preterm premature rupture of membranes, clinical chorioamnionitis, intrapartum death, abruption, complications of multiple gestation, and uterine rupture were excluded in the subset of stillbirths without obstetric complications.20
The delivery, defined as a case if there were any stillbirths delivered and as a control if all live births were delivered, was the unit of analysis. The analyses were weighted for oversampling and other aspects of the study design as well as for differential consent using SUDAAN 10.0 software.22 Construction of the weights has been previously described.12 Crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated from univariate and multivariable logistic regression models. All tests were performed at a nominal significance level of α=0.05. All single degree-of-freedom tests were two-sided.
The adjusted ORs account for stillbirth risk factors known at pregnancy confirmation (baseline) using a modification to a risk factor score for stillbirth that was developed on the logit scale using the coefficients from a logistic regression model. The model used data on all Stillbirth Collaborative Research Network deliveries where a maternal interview was conducted and a prenatal chart was abstracted. Variables contributing to the baseline risk 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 multiple gestation in current pregnancy as well as paternal age, family income, insurance and method of payment, and clinical site. All variables included in the score were categorical and a weighted average of the regression coefficients associated with the categories was used when a variable was missing for an observation. The weights were taken as the sample weighted proportion of live births by category. There were very few missing values, as previously noted.14 The modification to the risk factor score for this analysis was to exclude coefficients associated with pregnancy history.
Figure 1 depicts enrollment to the Stillbirth Collaborative Research Network and inclusion in this analysis. A total of 582 deliveries with stillbirth and 1,547 with live births had blood available and assessment of anticardiolipin and anti-β2-glycoprotein-I antibody levels. Women in the case group who did and did not enroll in the Stillbirth Collaborative Research Network were similar with regard to maternal age, maternal race and ethnicity, insurance and method of payment, and gestational age at delivery. Women in the control group who did not enroll differed from those who enrolled by maternal race and ethnicity and gestational age at delivery.21 The demographic characteristics of the Stillbirth Collaborative Research Network study participants have been published.14,21 Among women with stillbirth, those women refusing blood draw were more likely to be non-Hispanic black and nulliparous than those consenting (Table 1). Women with live births who were non-Hispanic black, younger than 20 years or 40 years and older, and did not receive early prenatal care were more likely to refuse blood draw (Table 1).
Abnormal levels of anticardiolipin and anti-β2-glycoprotein-I antibodies in all stillbirths compared with all live births are shown in Table 2. The proportion of deliveries with elevated levels of IgG anticardiolipin antibodies was higher in women in the case group compared with women in the control group (3.8 compared with 1.1%, OR 3.43, 95% CI 1.79–6.60). The same was true for IgG anti-β2-glycoprotein-I antibodies (OR 3.17, 95% CI 1.30–7.72). However, similar proportions of deliveries with stillbirth and with live births had elevated levels of IgM anticardiolipin and anti-β2-glycoprotein-I antibodies. The OR for at least one positive antibody test was 1.63 (95% CI 1.13–2.35). Results were similar for adjusted odds ratios (Table 2).
Table 2 also shows levels of anticardiolipin and anti-β2-glycoprotein-I antibodies in all stillbirths compared with term live births. Results were similar to the comparison between all stillbirths and all live births. Women having a stillbirth were more likely to have elevated levels of IgG anticardiolipin and anti-β2-glycoprotein-I antibodies than women having a term live birth.
Similar results also were noted when the subset of nonanomalous stillbirths was compared with term live births (Table 3). A greater percentage of women with nonanomalous stillbirths had elevated levels of IgG anticardiolipin antibodies (4.8%) compared with those with term live births (1.0%, OR 5.09, 95% CI 2.44–10.65). A positive test for IgG anti-β2-glycoprotein-I antibodies yielded an OR of 2.87 (95% CI 1.05–7.88) for stillbirth. Positive tests for IgM anticardiolipin and IgM anti-β2-glycoprotein-I antibodies were similar between groups. Having one or more positive test for an antibody was associated with a 2.02 OR (95% CI 1.34–3.05) for stillbirth.
Table 3 also depicts antibody results in women with stillbirth not associated with fetal anomalies or obstetric complications compared with women with term live births. IgG anticardiolipin antibodies were associated with a fivefold odds of stillbirth and IgM anticardiolipin antibodies were associated with a twofold odds of stillbirth. IgG anti-β2-glycoprotein-I antibodies were associated with a threefold odds of stillbirth, but IgM anti-β2-glycoprotein-I antibodies were not associated with stillbirth. Of these specific stillbirth deliveries, 11.7% had at least one positive test for antiphospholipid antibodies compared with 5.9% of those with term live births (OR 2.11, 95% CI 1.33–3.33).
We explored whether a threshold of 40 or more units of IgG or IgM anticardiolipin or anti-β2-glycoprotein-I antibodies was more strongly associated with stillbirth than using a threshold of 20 or more units. For each of the four antibodies and in each of the subgroups of stillbirth and live birth analyzed, a threshold of 40 units was not more strongly associated with stillbirth than a threshold of 20 units (data not shown).
We then assessed the clinical characteristics of the cases with positive tests for antiphospholipid antibodies in hopes of identifying specific scenarios that merit testing. Details for each case are shown in Table 4. In this study, there were 56 deliveries with stillbirth (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 antibodies. In these cases, a history of thrombosis was noted in one (1.8%) and none had systemic lupus erythematosus. Pregnancy complications included preeclampsia in 6 of 53 (11.3%), small-for-gestational-age fetus in 20 of 54 (37.0%), and clinical or histologic evidence of abruption in 14 of 56 (25.0%). Of the 50 stillbirths with a complete postmortem examination, seven (14%) had antiphospholipid antibody syndrome as a probable cause of death based on Initial Causes of Fetal Death criteria. It is noteworthy that many other cases in this subset of 57 stillbirths had a likely cause of death other than antiphospholipid antibody syndrome. One case of interest was congenital syphilis, which is known to cause antiphospholipid antibody production. In this cohort of 56 women with positive tests for antiphospholipid, 22 (39.3%) had no history of (prior) pregnancy loss, thrombosis, systemic lupus erythematosus, small-for-gestational-age fetus, or preeclampsia. Thus, we were unable to ascertain clinical features that would allow testing only a subset of patients with stillbirth for antiphospholipid.
Another important question is whether it is necessary to test for all four antibodies assessed in this study. Forty-six of the 56 (82.1%) women having a positive test result tested positive for only one of the four antibodies. Thus, not testing for any one of the antibodies would have potentially missed some cases. Only 19 patients with stillbirth had clinically indicated testing for lupus anticoagulant, and two of these were positive.
Elevated levels of IgG anticardiolipin and anti-β2-glycoprotein-I antibodies were associated with an approximate threefold increased odds of stillbirth when all stillbirth deliveries are compared with all live birth deliveries. However, levels of IgM anticardiolipin and anti-β2-glycoprotein-I antibodies were similar among groups. Women with stillbirth had an approximately 1.6-fold risk for having at least one positive antibody compared with those with live births.
When the subset of stillbirths not associated with fetal anomalies or obstetric complications was compared with term live births, elevated IgG anticardiolipin antibodies were associated with an OR of 5.30 (95% CI 2.39–11.76) for stillbirth. The OR for IgM anticardiolipin antibodies was 2.03 (95% CI 1.09–3.76) and the OR for IgG anti-β2-glycoprotein-I antibodies was 3.00 (95% CI 1.01–8.90). Stillbirth was not significantly associated with IgM anti-β2-glycoprotein-I antibodies. The OR for stillbirth among women with one or more positive antibody test compared with none was 2.11 (95% CI 1.33–3.33).
These data confirm and quantify the previously suspected association between antiphospholipid antibodies and stillbirth. A meta-analysis of 25 studies noted an OR for late recurrent fetal loss of 3.57 (95% CI 2.26–5.65) for any positive test for anticardiolipin antibodies.23 The OR for recurrent pregnancy loss in women with medium-to-high titer IgG anticardiolipin antibodies was 4.68 (95% CI 2.96–7.40).23 Although some women with stillbirth were included in these studies, most pregnancy losses were before 20 weeks of gestation, and all were less than 24 weeks of gestation.23
In addition, the women in the meta-analysis had recurrent rather than sporadic pregnancy loss. In contrast, our study is one of the few to address sporadic stillbirth. It is noteworthy that antiphospholipid antibodies are associated with recurrent early pregnancy loss23 but not sporadic early pregnancy loss.24 This is expected because sporadic early pregnancy loss is common and usually the result of genetic abnormalities. In contrast to early pregnancy loss, even a single fetal death after 20 weeks of gestation is considered to be clinical evidence of antiphospholipid antibody syndrome. Thus, our data support the Sapporo obstetric criteria for antiphospholipid antibody syndrome that include sporadic fetal death.
It is noteworthy that 56 of 582 (9.6%) women with stillbirth had positive tests for antiphospholipid antibodies. It is unclear that all of these women in the case group will prove to have antiphospholipid antibody syndrome because antibody levels in many cases were lower than those considered diagnostic of antiphospholipid antibody syndrome and they were not systematically assessed 12 weeks after the initial assay. Some cases had other potential causes of stillbirth, and many had only modestly elevated levels of antibodies. There were many cases in which the only clinical evidence of antiphospholipid antibody syndrome was unexplained stillbirth. Fourteen of these women in the case group had very high levels (greater than 40 units) of antibodies. Thus, it seems as though testing all women with unexplained stillbirth is a reasonable approach to the workup of stillbirth. Also, although IgG anticardiolipin antibodies were most strongly associated with stillbirth, testing for all four antibodies assessed in this study can identify additional women with potential antiphospholipid antibody syndrome compared with testing for IgG anticardiolipin antibodies alone.
It is important for clinicians to be aware that 6% of live births had at least one positive test for antiphospholipid antibodies. Thus, a positive test is not diagnostic of antiphospholipid antibody syndrome. It is critical to be sure that the stillbirth is otherwise unexplained when considering a diagnosis of antiphospholipid antibody syndrome. It is also imperative to repeat testing because positive tests may be transient. Finally, it should be understood that low positive test results (eg, levels between 20 units and 40 units) are not diagnostic of antiphospholipid antibody syndrome and that treatment is not proven to be efficacious in such cases.2
There were several weaknesses of our study. We did not assess lupus anticoagulant in each patient because we did not have available plasma. Second, women did not have serial assessment of antiphospholipid antibodies. These autoantibodies may fluctuate over time, and it is necessary to have elevated levels persist on two occasions at least 12 weeks apart to be considered diagnostic of antiphospholipid antibody syndrome.2 Third, we did not have detailed information regarding the possible influence of treatment for antiphospholipid antibody syndrome as we might be able to obtain in a longitudinal cohort study. Finally, the slight differences in women who did and did not agree to blood draw may limit the generalizability of our data.
The study also had numerous strengths. It is one of the few studies of antiphospholipid that was population-based, providing reliable estimates of the association of stillbirth with antiphospholipid antibodies. It also involved a geographically, racially, and ethnically diverse population, enhancing the generalizability of the results. In addition, it is one of the largest studies of antiphospholipid antibodies and stillbirth, including almost 600 women in the case group. Importantly, participants underwent an extensive evaluation for other potential causes of stillbirth. Finally, the laboratory performing the antiphospholipid antibody assays was blinded to clinical status of the samples.
In summary, elevated levels of IgG anticardiolipin and anti-β2-glycoprotein-I antibodies are associated with a threefold to fivefold increased odds of stillbirth. IgG anticardiolipin antibodies are more strongly associated with stillbirth than IgM antibodies. Almost 10% of participants with stillbirth had positive tests for antiphospholipid antibodies, and several had antiphospholipid antibody syndrome as a possible or probable cause of death. Our data support consideration of testing for antiphospholipid in cases of otherwise unexplained stillbirth.
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