Testing Levels of Anti-Ro/SSA Antibodies in Pregnant Patients to Identify Risk of Fetal Atrioventricular Block

Background: defining anti-Ro52 and anti-Ro60 antibody thresholds to identify unlikely development of f-AVB

A small percentage of pregnant patients with anti-Ro/SSA positive antibodies will carry a child who develops fetal atrioventricular block (f-AVB) during pregnancy. The strongest predictive risk factor is a previously affected child (18%); risk for first pregnancies or those with healthy offspring is 2%.

Fetal echocardiogram surveillance practices vary among institutions and have mostly not detected the transition from incomplete to irreversible complete f-AVB. Surveillance is also costly and time-consuming.

Previous evidence suggests lower anti-Ro/SSA antibody levels are linked to a reduced risk for f-AVB. There are few commercially available tests for anti-Ro52 and anti-Ro60 titers, both of which have long been associated with the development of f-AVB.

In this study, researchers from three institutions, including a lead author from the Fetal Cardiology Program in the Colorado Fetal Care Center at Children’s Hospital Colorado, sought to define anti-Ro52 and anti-Ro60 antibody thresholds to identify fetuses unlikely to develop AVB using clinically validated, commercially available and/or research laboratory tests.

Methods: an analysis of data from anti-Ro/SSA-positive pregnancies

The retrospective cohort study reviewed data from pregnant patients from three centers who tested positive in their local commercial laboratories for anti-Ro/SSA antibodies (anti-Ro52, anti-Ro60, or both).

Centers and timeline of records review:

• Children’s Colorado: 2014 to 2021
• Phoenix Children’s Hospital: 2014 to 2021
• New York University: 2002 to 2021

Patients were divided into three groups based on pregnancy outcomes:

• Group 1: Indicated no 1°, 2° or 3° f-AVB and primigravid or no history of f-AVB in previous pregnancy
• Group 2: Indicated 2° or 3° f-AVB in current pregnancy
• Group 3: Indicated no 2° or 3° f-AVB in current pregnancy but history of 2° or 3° f-AVB in previous pregnancy

In addition:

• Hydroxychloroquine use at time of pregnancy recorded for most patients
• Pregnancies with 1° f-AVB or isolated endocardial fibroelastosis excluded

Laboratory testing

Maternal sera were analyzed by Associated Regional and University Pathologists (ARUP) Laboratories using a clinically validated multiplex bead assay. Sera from pregnant NYU patients also were analyzed for a research enzyme-linked immunosorbent immunoassay.

• Only positive assays were included.
• All assays were performed without knowledge of the patients’ outcome group.

Statistical methods

This study calculated the negative predictive value separately for anti-Ro52 and anti-Ro60 antibodies and for the two combined using a logistic regression model and a parallel testing strategy.

Results: Testing both anti-Ro52 and anti-Ro60 titers can successfully identify low risk for f-AVB

Anti-Ro52 and anti-Ro60 antibody titers

• Maternal sera were obtained from 270 anti-Ro/SSA antibody-positive pregnancies:
  • 157 NYU
  • 113 Children’s Colorado and Phoenix Children’s
• Anti-Ro52 and anti-Ro60 titers were evaluated by:
  • 113 ARUP
  • 70 NYU
  • 87 both
• Groups
  • Group 1: 141 samples
  • Group 2: 66 samples
  • Group 3: 63 samples
• When compared to Group 1, researchers found Group 2 and Group 3 had:
  • Significantly higher median titers for ARUP anti-Ro52 and anti-Ro60
  • More similar anti-Ro/SSA antibody titers from both NYU and ARUP Laboratories

Hydroxychloroquine data

Models determining negative predictive value (NPV) for fetal atrioventricular block

Researchers compared three approaches using ARUP or NYU titers to target the goals of 100% NPV and maximizing the number of unaffected fetuses who would be correctly predicted as not developing AVB and able to forgo surveillance.

• Testing predictive value of anti-Ro52 alone
• Testing predictive value of anti-Ro60 alone
• Testing successful prediction of anti-Ro52 and anti-Ro60 together

ARUP negative predictive titers for fetal atrioventricular block

Two strategies performed well:

• Anti-Ro52 on its own with <44 AU/mL threshold performed well at maximizing proportion of healthy pregnancies
  • 51% pregnancies with no f-AVB
  • 0% f-AVB
• Anti-Ro 52 + 60 titers together resulted in joint 100% NPV <110 AU/mL threshold
  • 0% f-AVB
  • 50% pregnancies with no f-AVB

NYU negative predictive value for fetal atrioventricular block
NYU measurements yielded similar results to ARUP titers:

• Anti-Ro52 on its own with <440 EU threshold performed well at maximizing proportion of healthy pregnancies
  • 51% pregnancies with no f-AVB
  • 0% f-AVB
• Anti-Ro 52 + 60 titers together resulted in joint 100% NPV <650 and 4060 EU
  • 0% f-AVB
  • 53% pregnancies with no f-AVB

NYU and ARUP data subject comparison
Since only 28 patients from Groups 1 and 2 had both ARUP and NYU titers evaluated, researchers did not directly compare overall summaries.

Discussion: Detecting a threshold with 100% NPV for f-AVB is clinically important

Clinical implications from this study and others

• Anti-Ro/SSA antibody titers not sole predictors of f-AVB
• Recurrence rates not 100%
• Exposure up to highest levels of antibodies result in normal fetal conduction
• This study’s findings not unexpected
• Consider streamlined approach to surveillance, which excludes low titer subjects with no previous affected pregnancies
• Consider use and cost of serial echocardiogram surveillance for all anti-Ro/SSA antibody-positive pregnancies
• Identifying a threshold with 100% NPV for f-AVB is of great clinical importance

Research implications

• A clinical trial using this data is underway to risk stratify surveillance of anti-Ro/SSA antibody-positive pregnancies to only those pregnancies at risk of f-AVB.
• Hydroxychloroquine use does not affect anti-Ro/SSA antibody titers, but it does decrease the pathologic effects of the antibodies.

Conclusion: Testing for both anti-Ro52 and anti-Ro60 titers is optimal and could reduce serial echocardiograms

Researchers concluded that testing for both anti-Ro52 and anti-Ro60 titers is optimal because low values on both provide additional evidence suggesting low risk for fetal AVB. This also could reduce costly serial echocardiograms currently recommended for all anti-Ro/SSA-antibody positive pregnancies.