Antiretroviral therapy (ART) during pregnancy improves maternal health and reduces mother-to-child transmission of HIV to less than 2%1 in the United States. Such extensive ART use during pregnancy, however, requires careful monitoring for potential ART toxicity in prenatal HIV-exposed uninfected (HEU) children. The protease inhibitor atazanavir (ATV) is a preferred ART agent in pregnancy and is becoming one of the most commonly prescribed antiretroviral (ARV) drugs for pregnant women with HIV.1,2 Recent Pediatric HIV/AIDS Cohort Study (PHACS) research demonstrated associations between fetal ATV exposure and poor early language development risk.3,4 One-year olds whose mothers were prescribed ATV during pregnancy had lower mean Bayley Scales of Infant and Toddler Development—Third Edition5 (Bayley-III) Language domain scores compared with infants without ATV exposure.3 In a separate investigation, prenatal ATV-exposed 1-year olds also had increased late language emergence (LLE) risk but language screening at age 2 showed no significant association between LLE and ATV exposure.4 This association of intrauterine ATV exposure and lower performance on infant language development measures deserves further study.
Approximately 20% of maternal ATV in blood crosses the placenta, directly exposing the fetus.6 ATV also inhibits uridine diphosphate glucuronosyltransferase 1A1, the hepatic enzyme responsible for maternal and fetal bilirubin conjugation.7 However, as fetal liver bilirubin conjugation capacity is limited, the fetus relies primarily on maternal hepatic bilirubin conjugation to prevent neurotoxic bilirubin exposure to the vulnerable fetal brain.8,9 Neonatal unconjugated bilirubin exposure is associated with adverse neurologic and developmental outcomes, including language delays.10–13 Because maternal ATV administration leads to higher maternal unconjugated bilirubin concentrations,6 the fetus also may be exposed to elevated unconjugated bilirubin levels for a prolonged period.
Combination ART regimens are most effective at preventing mother-to-child transmission,14–16 but concomitant medications affecting maternal ATV pharmacokinetics may also affect fetal ATV exposure. Tenofovir disproxil fumarate (TDF) coadministration reduces ATV trough concentrations;17 similar ATV concentration effects were seen during pregnancy with third trimester TDF coadministration.18
Intrauterine ATV exposure may affect the fetus directly through transplacental drug transfer or indirectly through increased unconjugated bilirubin exposure; thus, accurate fetal ATV exposure quantification is critical to understanding infant and child outcomes. Previous fetal drug exposure research from terminated pregnancies demonstrated meconium, the first neonatal feces, begins to form early in the second trimester,19,20 with meconium drug concentrations reflecting drug exposure during the third and perhaps second trimesters.21,22 We recently developed a novel meconium ARV drug assay enabling quantitative in utero ARV exposure assessment.23 As previous studies reported associations between infant language development and intrauterine ATV exposure, we sought to quantify ARV meconium concentrations and investigate associations between ATV meconium concentrations, infant language measures, and maternal ATV medication history.
The prospective PHACS Surveillance Monitoring of ART Toxicities (SMARTT) study enrolls pregnant women with HIV and their infants at 22 US sites to evaluate long-term prenatal ART exposure effects.3 Infants enrolled between 22-weeks gestation and 1 week postnatal were included. Each site's institutional review boards approved the study, and written informed consent was obtained. ARV exposure information, including start and stop dates, was abstracted from medical charts.
Meconium ARV Quantification
Meconium was collected within 72 hours. Beginning in 2011, meconium was frozen immediately after collection; before 2011, meconium was refrigerated at study sites. Storage conditions were changed to ensure accurate analysis of alcohol use markers; unlike these other markers, meconium ATV concentrations proved equally stable under refrigerated and frozen conditions. Therefore, meconium ATV concentrations from both storage conditions were included. After laboratory receipt, all specimens were frozen ≤−20°C until analysis (0–6 years). Meconium ARV drugs were quantified by our validated liquid chromatography–tandem mass spectrometry method.23 Sixteen parent ARVs and 4 metabolites were quantified in 0.25 g meconium with 10–500 ng/g quantification limits.23 ATV linearity was 10–2500 ng/g; interassay imprecision and accuracy were 3%–5% and 85%–119%, respectively.23
The Bayley-III3 Language domain provides an age-referenced standardized measure of language development from 1 to 42 months (mean score, SD; 100 ± 15). The MacArthur–Bates Communicative Development Inventory (CDI)4 provides gender-specific age-adjusted percentile scores in 4 domains: Phrases Understood, Vocabulary Comprehension, Word Production, and Total Gestures. Validity of each Bayley-III assessment was determined by local examining psychologists; when needed, assessment results were reviewed by a study team member to resolve questions. When several CDI questionnaire items were omitted, scores were reviewed by a study team language expert. Both measures were administered at 9–15 months (the 1-year study visit). Bayley-III scales were administered directly to infants; the CDI was administered as a parent/caregiver interview using the age-appropriate CDI Words and Gestures form. The Bayley-III is available only in English, whereas the CDI is available in English and Spanish. For this study, LLE was defined as a CDI score ≤10th percentile in 1 or more of the 4 domains.
ATV meconium concentration and language outcome distributions were inspected and appropriate transformations performed to achieve approximate normal distributions. Spearman correlations (ρ) of meconium ATV concentration with ATV exposure duration and timing were calculated. Infants whose mothers had interrupted ATV use during pregnancy (>3 days gap between 2 regimens or stopped ATV use before delivery) were excluded from certain analyses because maternal time off ATV can affect meconium drug concentrations. ATV meconium concentrations from infants whose mothers stopped ATV use before delivery were compared separately with those with uninterrupted intrauterine ATV exposure with the Wilcoxon rank sum test. A Jonckheere–Terpstra test assessed the trend between ATV meconium concentration and prenatal ATV exposure timing.
General linear and logistic regression models were built for continuous and binary language measures, respectively. Univariable analyses first identified potential confounders. For each outcome, a core model was obtained by identifying covariates associated with the outcome with P < 0.20 in univariable models and retained with P < 0.10 in multivariable models. Additionally, when an ATV exposure was added to the model, covariates not included initially were evaluated and those that changed ATV exposure estimates by at least 10% and were associated with the outcome (P < 0.10) were included. Multivariable models were then built to estimate associations of adjusted ATV exposure with language outcomes, controlling for all identified covariates. Concomitant TDF was forced in multivariable models a priori. Maternal and infant characteristics evaluated as potential covariates are described in Table 1. Maternal CD4 and HIV RNA during pregnancy were excluded from adjusted models; most measurements were obtained after ATV initiation, and therefore, we cannot control for medical reasons that may have indicated why a mother started ATV. These measurements could potentially be confounders or intermediates on the causal pathway between ATV exposure and language outcomes.
To evaluate maternal concomitant TDF impact on meconium ATV concentrations, we compared ATV concentrations from infants exposed to uninterrupted TDF + ATV + ritonavir with those from infants exposed to uninterrupted ATV + ritonavir with Wilcoxon rank sum test. Group language measures were compared by a 2-sample t test or Fisher exact test, as appropriate.
Sensitivity analyses evaluated the impact of adjusting for premature birth and low birth weight, covariates possibly on the causal pathway between ATV exposure and language outcomes.5 Sensitivity analyses also adjusted for research site differences, using generalized estimating equation models considering individual language evaluations within sites as repeated measures. Unpaired t test, χ2 test, and Fisher exact test compared language scores and neonatal prophylaxis with previously published samples.
Of 1817 SMARTT Dynamic cohort infants enrolled through January 1, 2014, 432 (24%) were exposed to ATV during pregnancy. Meconium specimens were available for ARV quantification from 175 of these infants. Supplemental Table 1 (see Supplemental Digital Content, http://links.lww.com/QAI/A639) compares maternal and infant demographic characteristics between these 175 and the other 257 infants. Demographic characteristics were similar between groups with a few exceptions; infants with meconium ATV results were less likely to be Black, preterm, have low birth weight or caregivers with English as their primary language, and were more likely to have longer cumulative ATV exposure durations. Of our 175 ATV-exposed infants with meconium available, 93 had valid Bayley-III Language scores and 106 had valid CDI scores. Table 1 shows our study samples' maternal and infant demographic characteristics. The percentage of mothers with viral suppression (HIV RNA ≤400 copies per milliliter) increased from 48%–53% at the earliest to 84%–86% at the latest measure in pregnancy, whereas the percentage with CD4 ≥350 cells per cubic millimeter showed smaller increases from 70%–72% at the earliest to 75%–79% at later measures. Maternal substance use in pregnancy was similar to SMARTT overall.25 Most infants in our analysis were born in 2010 or 2011. No infant demonstrated hearing loss, based on hearing screening or caregiver report.
Of the 175 meconium specimens with ARV quantification, 166 infants had uninterrupted ATV exposure (≤3 days gap) in the second and third trimesters. Five mothers discontinued ATV before delivery; their infants' meconium ATV concentrations were considered separately when uninterrupted exposure was required. Four mothers had a more than 3-day interruption in their ATV-containing regimen during the second or third trimester but returned to an ATV regimen predelivery; these infants were excluded from all analyses requiring uninterrupted exposure.
Maternal ATV Duration and Meconium ATV
Median (range) meconium ATV concentration in the 166 infants with uninterrupted second and third trimester ATV exposure was 16,929 ng/g (29–143,018). Many women were on ATV throughout pregnancy; median (range) uninterrupted ATV exposure duration, excluding first trimester ATV administration, was 24 weeks (2.6–28.1). Uninterrupted ATV exposure duration was positively correlated with ATV meconium concentrations (ρ = 0.230, P = 0.003).
Maternal ATV Initiation and Meconium ATV
Most women using ATV during pregnancy started ATV before pregnancy or in the first trimester. Gestational ATV initiation week for women who started ATV before pregnancy or in the first trimester was left truncated to exclude the first trimester and set to 14.1 weeks, as meconium only begins to form at the beginning of the second trimester. Median (range) ATV initiation week during pregnancy was 14.1 (14.1–35.7). Meconium ATV concentrations were grouped by ATV initiation week (in or before early second trimester, ≤21 weeks; in late second, 21.1–28 weeks; early third, 28.1–34 weeks; or late third trimester, 34.1–42 weeks), revealing a trend toward decreasing median ATV meconium concentrations with later maternal ATV initiation (P = 0.07, Table 2). ATV initiation week negatively correlated with ATV meconium concentration (ρ = −0.215, P = 0.01).
Five infants whose mothers discontinued ATV-containing regimens before delivery had significantly lower ATV meconium concentrations than the 166 infants whose mothers remained on ATV through delivery (P < 0.001). These 5 mothers discontinued ATV 19–64 days before delivery, their ATV duration before cessation ranged from 4.1 to 21.6 weeks, and infant meconium ATV concentrations were 102–3468 ng/g. These 5 infants' gestational age at birth was 37–40 weeks and at the time ATV was discontinued 28.9–35.3 weeks.
Language Measures and Meconium ATV Concentrations
Among the 93 infants with Bayley-III data, the median (interquartile range) Language composite score was 94 (86–97) and ATV meconium concentrations ranged from 48 to 78,963 ng/g. Infant's cumulative ATV exposure duration, over the entire pregnancy, ranged from 4.1 to 42 weeks. Higher Bayley-III Language scores were associated with longer cumulative ATV exposure duration (see Supplemental Table 2, Supplemental Digital Content, http://links.lww.com/QAI/A639 and Table 3). Meconium ATV concentrations were not associated with Bayley-III Language scores in unadjusted or adjusted models; additionally, mean Bayley-III scores were not significantly different between infants with the lowest (≤10th percentile) and highest (>90th percentile) ATV meconium concentrations.
Among the 106 infants with CDI data, median (interquartile range) percentile scores were Vocabulary Comprehension, 45 (20–70); Word Production, 50 (33–60); Phrases Understood, 60 (35–75); and Total Gestures, 52.5 (30–70), and mean CDI percentile score was across all domains, 50 (34.8–63.8). ATV meconium concentrations ranged from 48 to 85,166 ng/g and cumulative ATV duration from 4.1 to 41.6 weeks. Higher CDI Phrases Understood scores were associated with longer ATV durations, both for cumulative and uninterrupted ATV duration measures (Table 3). Meconium ATV concentrations were not associated with individual CDI domains or average CDI percentile scores (see Supplemental Table 2, Supplemental Digital Content, http://links.lww.com/QAI/A639 and Table 3). Mean CDI scores were not significantly different between infants with the lowest and highest decile ATV meconium concentrations.
Most (92%) CDI-evaluated infants received zidovudine-only neonatal prophylaxis; 8 infants received zidovudine with other drugs (nevirapine, stavudine, or lamivudine + stavudine + nevirapine). Prophylaxis duration ranged from 1 to 66 days. There was no difference in the proportion of infants who received combination prophylaxis among those with LLE risk (4/25) and those without LLE risk (4/81, P = 0.09); additionally, neonatal prophylaxis duration was not significantly different between the 2 groups.
Maternal Concomitant ART Medication
Most mothers (77% of the 166 with uninterrupted ATV exposure) were on a TDF-containing ATV regimen. Three infants exposed to other ATV regimens were excluded from this comparison. Figure 1 illustrates group differences by uninterrupted ATV exposure duration. (Fig. 1). Bayley-III and CDI scores and LLE risk prevalence were not significantly different between infants exposed to TDF + ATV + ritonavir and ATV + ritonavir. The most prevalent maternal combination ART regimen in our sample was TDF + emtricitabine + ritonavir + ATV followed by zidovudine + lamivudine + ritonavir + ATV.
Similar results as the primary analyses were observed from sensitivity analyses. Adjusting for preterm birth and low birth weight or accounting for within-site correlations did not change the associations except significant associations of cumulative ATV exposure duration with Bayley-III Language and CDI Phrases Understood scores were attenuated after accounting for differences within sites.
Mean Bayley-III Language scores obtained in this study (93.2 ± 10.7) were lower than mean scores from the Bayley-III standardization sample24 (P < 0.001). Infants in our study also had significantly higher mean Bayley-III Language scores, higher CDI Total Gestures percentile scores, and lower LLE risk incidence compared with those in previously published SMARTT studies,3,4 excluding infants who contributed data to both (Bayley-III, n = 16; CDI, n = 30). The mean Bayley-III Language score from our unique infant cohort (n = 77) was 93.1 (±10.7) compared with the mean, 88.8 (±14.2), of unique infants with ATV exposure (n = 62) in the previously published cohort (P = 0.04). Our unique CDI-evaluated cohort (n = 76) obtained a higher Total Gestures domain percentile score compared with those in the previous report (53 versus 35, P = 0.02); otherwise, there were no differences between cohorts across CDI domains. LLE risk incidence among infants with ATV exposure in our sample was lower than in the previous report (22% versus 37%, P = 0.05).4 Proportion of infants who received combination neonatal prophylaxis and duration of this treatment among those with and without LLE risk were not significantly different between the 2 unique ATV-exposed groups.4
In utero HIV-infected exposure was previously associated with increased language impairment risk among 7- to 16-year olds with prenatal HIV exposure (HIV infected, 51%; HEU, 37%) compared with the general US population (16%).28 Associations at 1 year of age between prenatal ATV exposure, lower Bayley-III Language scores,3 and elevated LLE risk4 also were identified. As ATV use during pregnancy becomes more widespread,1,2 these associations warrant further monitoring.
In this investigation, longer ATV exposure durations were associated with higher ATV meconium concentrations, which were protective against LLE risk. Confirming this association was our finding that longer cumulative ATV exposure durations resulted in higher Bayley-III Language scores. These results differ from 2 previous SMARTT reports,3,4 possibly because of sampling differences associated with small samples or random variability. There was minimal overlap between the samples of the 3 studies; only 30 of our 106 CDI-evaluated infants and 16 of our 93 Bayley-evaluated infants were included in previous SMARTT studies. Infants in our study also had lower LLE risk and higher Bayley-III Language scores compared with those in our previous research; these improved outcomes may have challenged confirmation of previous negative ATV exposure associations or may be causative for the decreased LLE risk observed here compared with the previously seen increased LLE risk. Additionally, our results confirm previous PHACS research that showed increased LLE risk among HEU children; 24%–42% of HEU children in our study and previous PHACS studies4,28 demonstrated risk of LLE or language impairment compared with an expected prevalence of 16%–20% among children in the general population.28
Changing ARV use during pregnancy trends, random sampling differences, and cohort effects may have contributed to differences among the studies. Healthier women are receiving ATV more often now than in previous years; additionally, pregnant women with HIV may be healthier now because of continuous use of well-tolerated medications with lower pill burdens.1,2 Most infants included in this investigation were born later than infants in the previous SMARTT analyses; 78% of infants in the current study with Bayley-III data and 59% with CDI data were born after the cutoff dates of earlier analyses (January 5, 2009, and January 4, 2010, respectively).3,4 Further investigations must clarify these different results; however, the present findings confirm ATV exposure safety on infant language development, which supports the 2014 Department of Health and Human Services recommendations1 of ritonavir-boosted ATV as a preferred ART regimen for pregnant women with HIV. Further study also should determine if associations remain consistent as children age.
A potential mechanism for our observed lowered LLE risk may be ATV clearance into meconium. Higher ATV meconium concentrations are likely an indication of fetal ATV detoxification. ATV is primarily eliminated via hepatic pathways.29 In adults, ATV is metabolized by CYP3A4 and CYP3A5,30,31 mainly by oxidation, although several metabolites and metabolic pathways are known.32,33 ATV metabolism varies widely as adults on steady-state ritonavir-boosted ATV showed cumulative metabolite concentrations from 4% to 32% of parent ATV concentrations.33 Fetal ATV metabolism and clearance are unknown; however, published fetal liver CYP3A studies indicate fetal ATV metabolism is limited. Fetal CYP3A4 content and activity are <10% of adult levels,34–36 and little is known about fetal CYP3A5 activity.35,36 CYP3A7 accounts for 87%–100% of total fetal liver CYP3A content and shows high catalytic activity toward endogenous steroids but lower activity toward exogenous substances, traditional CYP3A4 substrates.37,38 Our data suggest a potential mechanism for the lowered LLE risk via fetal ATV clearance capacity, although ATV clearance to meconium also may be affected by maternal and infant factors not investigated in this study.
Associations between infant meconium ATV concentrations, uninterrupted second and third trimester ATV exposure duration, and gestational ATV initiation week were significant. Meconium drug concentrations can be influenced by variations in maternal dosage, fetal exposure duration and timing, placental transfer, and maternal and fetal pharmacokinetics and metabolism. Our significant observations and wide ATV meconium concentration range among women with uninterrupted ATV exposure (29–143,018 ng/g) demonstrate the significance of these influencing factors.
Three potentially reactive CYP3A4-generated ATV metabolites (an aromatic aldehyde, alpha-hydroxyaldehyde, and hydrazine metabolite) were recently identified in human liver microsome incubations.39 Oral ATV administration to mice, however, produced no detectable urinary or fecal concentrations of these metabolites.39 Potential metabolite toxicity is not yet known, though similar chemical structures are associated with glutathione and protein adducts, hepatic lesions, and neurotoxicity.39–41 Initially, we hypothesized that higher ATV meconium concentrations might indicate greater fetal exposure and therefore predict lower language scores and higher LLE risk. Our findings now suggest that higher ATV meconium concentrations likely indicate greater fetal ATV detoxification as there is less ATV available to form potentially toxic metabolites and hence better language outcomes. Additionally, lower ATV meconium concentrations suggest reduced ATV clearance, likely associated with higher fetal plasma ATV or metabolite concentrations throughout pregnancy, which might be correlated with increased LLE risk. Human plasma and meconium metabolite concentrations should be investigated, although commercial standards are currently unavailable. Further study into these metabolites' clinical implications is needed. However, as ritonavir is commonly coadministered, the in vitro metabolite study failed to investigate ritonavir's reduction of potentially toxic ATV metabolite formation, as seen with lopinavir.42
Associations between infant meconium ATV concentrations, uninterrupted second and third trimester ATV exposure duration, and gestational ATV initiation week were significant. Longer ATV exposure periods correlated with higher ATV meconium concentrations, and later ATV initiation resulted in lower ATV meconium concentrations. These correlations were weak (ρ < ±0.25), suggesting meconium ATV concentrations do not predict and only partially reflect maternal ATV duration or initiation timing.
TDF addition to a mother's ritonavir-boosted ATV therapy resulted in a decreasing meconium ATV concentration trend, as predicted from previous pharmacokinetic studies.18 However, no group language differences were observed between infants exposed to ritonavir-boosted ATV regimens with and without TDF.
Although our investigation was unique, it was limited by lack of information about neonatal and maternal bilirubin concentrations, maternal uridine diphosphate glucuronosyltransferase 1A1 polymorphisms, and infant phototherapy. Future studies could use these additional tests to better understand factors contributing to ATV's impact on infant language development. Further investigation of pathophysiological and psychosocial influences on HEU infant language development is needed to develop targeted interventions to address potential language delays. Previous research indicated that socioeconomic factors, genetic influences, and maternal HIV disease status may affect language acquisition.4,28,43
ATV meconium quantification provided a novel in utero ATV exposure assessment and, for the first time, demonstrated the value of including meconium ARV concentrations in longitudinal research on HIV. Higher meconium ATV concentrations were protective of LLE risk. Clinically, this information supports ATV safety during pregnancy for infant language development. Additionally, higher ATV meconium concentrations observed in our study may indicate greater fetal ATV detoxification as these concentrations protected against LLE risk. With ATV now a preferred ARV for pregnant women with HIV, our data add evidence to fetal ATV exposure safety for language development. Our ATV meconium research findings will be helpful to medical professionals evaluating in utero ATV exposure safety for infant language development.
The authors thank the children and families for their participation in PHACS and the individuals and institutions involved in the conduct of PHACS. Data management services were provided by Frontier Science and Technology Research Foundation (Principal Investigator: Suzanne Siminski), and regulatory services and logistical support were provided by Westat, Inc (Principal Investigator: Julie Davidson).
The following institutions, clinical site investigators, and staff participated in conducting PHACS SMARTT in 2012, in alphabetical order: Baylor College of Medicine: William Shearer, Mary Paul, Norma Cooper, and Lynette Harris; Bronx Lebanon Hospital Center: Murli Purswani, Emma Stuard, and Anna Cintron; Children's Diagnostic and Treatment Center: Ana Puga, Dia Cooley, Doyle Patton, and Deyana Leon; Ann and Robert H. Lurie Children's Hospital of Chicago: Ram Yogev, Margaret Ann Sanders, Kathleen Malee, and Scott Hunter; NY University School of Medicine: William Borkowsky, Sandra Deygoo, and Helen Rozelman; St Jude Children's Research Hospital: Katherine Knapp, Kim Allison, and Megan Wilkins; San Juan Hospital/Department of Pediatrics: Midnela Acevedo-Flores, Lourdes Angeli-Nieves, and Vivian Olivera; SUNY Downstate Medical Center: Hermann Mendez, Ava Dennie, and Susan Bewley; Tulane University Health Sciences Center: Russell Van Dyke, Karen Craig, and Patricia Sirois; University of Alabama, Birmingham: Marilyn Crain, Newana Beatty, and Dan Marullo; University of California, San Diego: Stephen Spector, Jean Manning, and Sharon Nichols; University of Colorado Denver Health Sciences Center: Elizabeth McFarland, Emily Barr, and Robin McEvoy; University of Florida/Jacksonville: Mobeen Rathore, Kristi Stowers, and Ann Usitalo; University of Illinois, Chicago: Kenneth Rich, Lourdes Richardson, Delmyra Turpin, and Renee Smith; University of Medicine and Dentistry of New Jersey: Arry Dieudonne, Linda Bettica, and Susan Adubato; University of Miami: Gwendolyn Scott, Claudia Florez, and Elizabeth Willen; University of Southern California: Toinette Frederick, Mariam Davtyan, and Maribel Mejia; University of Puerto Rico Medical Center: Zoe Rodriguez, Ibet Heyer, and Nydia Scalley Trifilio.
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