Abstract
Combination antiretroviral therapy (cART) during pregnancy prevents vertical transmission, but many antiretrovirals cross the placenta and several can affect mitochondria. Exposure to maternal human immunodeficiency virus (HIV) and/or cART could have long-term effects on children who are HIV exposed and uninfected (CHEU). Our objective was to compare blood mitochondrial DNA (mtDNA) content in CHEU and children who are HIV unexposed and uninfected (CHUU), at birth and in early life.
Whole-blood mtDNA content at birth and in early life (age 0–3 years) was compared cross-sectionally between CHEU and CHUU. Longitudinal changes in mtDNA content among CHEU was also evaluated.
At birth, CHEU status and younger gestational age were associated with higher mtDNA content. These remained independently associated with mtDNA content in multivariable analyses, whether considering all infants, or only those born at term. Longitudinally, CHEU mtDNA levels remained unchanged during the first 6 months of life, and gradually declined thereafter. A separate age- and sex-matched cross-sectional analysis (in 214 CHEU and 214 CHUU) illustrates that the difference in mtDNA between the groups remains detectable throughout the first 3 years of life.
The persistently elevated blood mtDNA content observed among CHEU represents a long-term effect, possibly resulting from in utero stresses related to maternal HIV and/or cART. The clinical impact of altered mtDNA levels is unclear.
The number of children who are human immunodeficiency virus (HIV) exposed and uninfected (CHEU) is increasing worldwide, and most women living with HIV now conceive while already on combination antiretroviral (ARV) therapy (cART) [1]. Although the benefits of cART during pregnancy are unquestionable, concerns remain regarding possible long-term adverse effects of ARV exposure. In addition to adverse outcomes, such as stillbirth, preterm birth, small size for gestational age, and intrauterine growth restriction [2–4], CHEU exposed to ARVs during gestation are at increased risk of immune system abnormalities [5, 6], hyperlactatemia [7], and metabolic complications [8]. Some studies have also reported neurodevelopmental and language delays among CHEU [9–12]; however, others did not [13, 14]. Many of these conditions could be related to mitochondrial dysfunction, which itself may be reflected by altered mitochondrial DNA (mtDNA) levels.
In the general population, mtDNA levels are a marker of cellular health, and both elevated and decreased levels have been implicated in many age-related comorbid conditions [15]. Several studies have reported increased mitochondrial dysfunction among CHEU who had in utero exposure to nucleoside reverse-transcriptase inhibitors (NRTIs) [16–19]. These can inhibit mtDNA polymerase γ and alter mtDNA levels [20, 21]. Older NRTIs induced mitochondrial toxicities and decreased mtDNA levels [21–24], an effect that appeared at least partially reversible, because mtDNA levels rebounded on discontinuation of therapy [21]. Most NRTIs readily cross the placenta [25, 26] and could affect the developing fetus.
Brogly et al [19] reported lower mtDNA levels in peripheral blood mononuclear cells of ARV-exposed CHEU with or without mitochondrial dysfunction. Some cross-sectional studies have also reported lower mtDNA levels in CHEU [27–31], whereas others reported increased mtDNA content in CHEU at birth and during early life [32–34]. Budd et al [35] described elevated blood mtDNA content in older CHEU diagnosed with autism spectrum disorder. Changes in ARV regimens may explain some of the variability between studies, many of which were also limited by sample size and/or characteristics of their control groups. To address these inconsistencies, our objective was to compare blood mtDNA content at birth in a larger cohort of CHEU and children who are HIV unexposed and uninfected (CHUU) and to investigate potential relationships with in utero exposure to maternal cART. We hypothesized that mtDNA content would be altered at birth and associated with certain maternal cART regimens. We also report on the longitudinal changes in CHEU mtDNA content over the first 3 years of life.
METHODS
Study Sample
Our study sample consisted of the same cohort of CHEU and CHUU <3 years of age, for which we recently reported leukocyte telomere length at birth and in early life [36] and used the same DNA extracts. Briefly, children were enrolled in 3 Canadian cohort studies: (1) a pediatric cohort of children born between 2003 and 2006 in Vancouver, British Columbia, or Toronto, Ontario; (2) a pregnancy cohort (infants born between 2005 and 2009 in Vancouver); and (3) the Children and Women: AntiRetrovirals and Markers of Aging (CARMA) cohort (born between 2006 and 2012 in Vancouver, Toronto, Ottawa [Ontario], or Montreal [Quebec]) [36]. Supplementary Figure 1 and Supplementary Table 1 provide information on the study design, the study sample, and the 3 cohorts, including inclusion and exclusion criteria. In addition, 50% of CHUU were anonymous, as we used leftover blood from routine tests; only sex and age were known. A subset of children (n = 154 (24%); 73 CHEU and 81 CHUU), all in the pediatric cohort, were previously examined [32] and included again here, to ensure a wide breadth of in utero ARV exposure with respect to type and duration of cART. Their specimens were reassayed using a different quantitative polymerase chain reaction technique. Approximately two-thirds of CHEU participants had >1 blood specimen collected between birth and age 3 years, while each CHUU provided a single blood specimen. All participants with a birth specimen were from Vancouver.
The study was approved by the institutional review boards and informed consent was obtained unless not required (see Supplementary Table 1 footnote). Demographic and clinical data, including exposures during pregnancy were obtained from cohort databases. Maternal ethnicity and smoking during pregnancy were self-reported. Owing to heterogeneity in smoking data collection across cohorts, we categorized this variable as tobacco smoking ever during pregnancy (yes vs no), irrespective of the intensity, frequency, or duration of smoking in pregnancy. Children born at <37 weeks of gestation and those with a birth weight below the 10th percentile relative to Canadian neonates of the same gestational age [37] were defined as preterm and small for gestational age, respectively.
mtDNA Content Measurement
Whole-blood (WB) mtDNA content was expressed as a ratio between mtDNA and nuclear DNA copies, measured via monochrome multiplex different quantitative polymerase chain reaction [38]. We assayed all specimens from an individual CHEU participant on the same plate; each plate contained both specimens from both CHEU and CHUU, randomized. The intra-assay and interassay variability were 7.9% and 8.4%, respectively. We excluded specimens not meeting quality control criteria of <15% difference between replicates over 2 attempts (Supplementary Figure 1) [38].
Statistical Analyses
MtDNA content values were log10-transformed to achieve normal (CHUU) or near-normal (CHEU) distributions. Clinical and demographic characteristics of CHEU and CHUU were compared using Student t or Mann-Whitney U tests for continuous variables, and χ 2 tests for categorical variables. Spearman correlations were used to investigate univariate associations between log10 mtDNA content at birth and gestational age at birth, birth weight, maternal age at delivery, and duration of in utero cART exposure. We used t or Mann-Whitney tests to assess associations between mtDNA content at birth and the following: HIV exposure status, infant sex, preterm birth, small size for gestational age, and smoking ever during pregnancy. We also compared mtDNA content at birth between various maternal ethnicities and cART regimens using Kruskal-Wallis or analysis of variance tests, with Dunn or Tukey tests to adjust for multiple pairwise comparisons. Factors important univariately (P < .10) were considered in multivariable analyses of covariance. The final models were constructed in a stepwise manner, by minimizing the Akaike information criteria. Collinearity between variables was assessed, and, if present, variables were included in the model in turn.
To determine the relationship between WB mtDNA content and age during the first 3 years of life, a subset of distinct CHEU and CHUU were sex and age matched 1:1 (±2 days within the first 2 weeks; ±8 days from 2 weeks to 1 year; ±15 days from 1–3 years). We then compared the slopes and intercepts of the 2 groups’ linear regressions, using GraphPad Prism V8 software. We also used generalized additive mixed-effects models or linear mixed-effects models to analyze the longitudinal trajectory of mtDNA content among all CHEU, with the mgcv package in R v.3.4.2 software. Finally, comparisons of mtDNA content between birth and the closest subsequent visit during the prophylaxis period were performed using paired t tests.
RESULTS
Characteristics of Study Participants
All Participants
A total of 324 CHEU had ≥1 WB specimen available for this study, collected between birth and age 3 years. Of these, 114 (35%) had a birth specimen, and 214 (66%) had ≥2 longitudinal specimens. For CHUU, 306 had a single WB specimen each; 88 (29%) of these were collected at birth, and 154 (50%) were anonymous controls. Infant and maternal characteristics are detailed in Table 1. Among CHEU and CHUU for whom this information was available, maternal age, history of smoking during pregnancy, infant sex, gestational age at birth, birth weight, preterm status, Apgar scores, and rates of small size for gestational age were similar. However, they differed significantly in maternal ethnicity, with approximately 50% of CHEU compared with <1% of CHUU born to African Caribbean black women (P < .001).
Demographic and Clinical Characteristics of CHEU and CHUU, Including All Participants and Children With a Blood Specimen at Birtha
| Characteristics | All Participants, No. (%)b | Children With a Blood Specimen at Birth, No. (%)b | ||||
|---|---|---|---|---|---|---|
| CHEU (n = 324) | CHUU (n = 306) | P Value | CHEU (n = 114) | CHUU (n = 88) | P Value | |
| Infant characteristics | ||||||
| Male sex | 161 (50) | 169 (55) | .16 | 64 (56) | 47 (53) | .70 |
| Gestational age, median (range) [IQR], wk | 38.4 (27.1–41.7) [37.2–39.6] (n = 323) | 39.0 (28.3–42.1) [37.4–40.1] (n = 149) | .07 | 38.3 (31.3–41.6) [37.3–39.6] | 39.4 (28.9– 42.1) [38.2–40.3] | <.001 |
| Preterm delivery (<37 wk) | 61 (19) | 28 (19) | 23 (20) | 9 (10) | ||
| <37 and ≥34 wk | 42 (13) | 17 (11) | .98 | 19 (17) | 8 (9) | .055 |
| <34 wk | 19 (6) | 11 (7) | 4 (4) | 1 (1) | ||
| Birth weight, median (range) [IQR], kg | 3.1 (1.6–4.2) [2.7–3.4] (n = 184) | 3.2 (1.2–5.2) [2.7–3.6] (n = 150) | .32 | 3.1 (1.6–4.1) [2.7–3.4] | 3.4 (1.4–5.2) [3.0–3.7] | <.001 |
| SGA | 30 (16) (n = 184) | 27 (18) (n = 149) | .66 | 19 (17) | 12 (14) | .55 |
| Apgar score at 5 min, median (range) [IQR] | 9 (5–10) [9–9] (n = 181) | 9 (3–10) [9–9] (n = 138) | .94 | 9 (5–10) [9–9] (n = 113) | 9 (3–10) [9–9] (n = 87) | .56 |
| Maternal characteristics | ||||||
| Maternal age at delivery, median (range) [IQR], y | 31.5 (16.7–45.3) [27.1–35.5] | 32.2 (16.7–44.0) [28.7–35.7] (n = 135) | .15 | 31.0 (17.4– 42.4) [26.8–35.2] | 32.5 (21.3– 43.0) [29.8–35.8] | .06 |
| Maternal ethnicity | ||||||
| Indigenous | 42 (13) | 9 (3) | <.001 | 33 (29) | 7 (8) | <.001 |
| African Caribbean black | 158 (49) | 2 (0.7) | 28 (25) | 2 (2) | ||
| White | 84 (26) | 93 (30) | 39 (34) | 55 (63) | ||
| Asian | 21 (6) | 29 (9) | 10 (9) | 16 (18) | ||
| Other/unknown | 12/7 (6) | 19/154 (57) | 4/0 (4) | 1/7 (9) | ||
| Maternal smoking ever in pregnancy | 105 (33) (n = 322) | 45 (30) (n = 152) | .51 | 63 (55) | 38 (43) | .09 |
| Detectable HIV pVL (>50 copies/mL) close to delivery | 18 (12) (n = 145) | NA | … | 12 (12) (n = 99) | NA | … |
| cART characteristics | ||||||
| Duration of in utero cART exposure, median (range) [IQR], wk | 24.4 (0.0– 41.7) [16.6–37.4] (n = 322) | NA | … | 20.6 (0.0–41.1) [14.8–36.6] | NA | … |
| Maternal cART initiation | ||||||
| Before conception | 124 (38) | NA | … | 40 (35) | NA | … |
| In 1st trimester | 39 (12) | NA | 15 (13) | NA | ||
| In 2nd trimester | 124 (38) | NA | 46 (40) | NA | ||
| In 3rd trimester | 29 (9) | NA | 13 (11) | NA | ||
| Naive/unknown | 7/1 (2) | NA | 0 (0) | NA | ||
| Maternal cART regimen | ||||||
| AZT + 3TC + NVP | 26 (8) | NA | … | 7 (6) | NA | … |
| AZT + 3TC + NFV | 83 (26) | NA | 34 (30) | NA | ||
| AZT + 3TC + LPV/r | 112 (35) | NA | 45 (39) | NA | ||
| ABC + 3TC + PI/r | 32 (10) | NA | 10 (9) | NA | ||
| TDF + FTC (or 3TC) + PI/r | 27 (8) | NA | 10 (9) | NA | ||
| Otherc | 36 (11) | NA | 8 (7) | NA | ||
| Naive/unknown | 7/1 (2) | NA | 0 (0) | NA | ||
| Infant AZT prophylaxis, median (range) [IQR], wk | 6 (0–8) [5–6] (n = 323)d | NA | … | 5 (2–6) [4–6] | NA | … |
| Characteristics | All Participants, No. (%)b | Children With a Blood Specimen at Birth, No. (%)b | ||||
|---|---|---|---|---|---|---|
| CHEU (n = 324) | CHUU (n = 306) | P Value | CHEU (n = 114) | CHUU (n = 88) | P Value | |
| Infant characteristics | ||||||
| Male sex | 161 (50) | 169 (55) | .16 | 64 (56) | 47 (53) | .70 |
| Gestational age, median (range) [IQR], wk | 38.4 (27.1–41.7) [37.2–39.6] (n = 323) | 39.0 (28.3–42.1) [37.4–40.1] (n = 149) | .07 | 38.3 (31.3–41.6) [37.3–39.6] | 39.4 (28.9– 42.1) [38.2–40.3] | <.001 |
| Preterm delivery (<37 wk) | 61 (19) | 28 (19) | 23 (20) | 9 (10) | ||
| <37 and ≥34 wk | 42 (13) | 17 (11) | .98 | 19 (17) | 8 (9) | .055 |
| <34 wk | 19 (6) | 11 (7) | 4 (4) | 1 (1) | ||
| Birth weight, median (range) [IQR], kg | 3.1 (1.6–4.2) [2.7–3.4] (n = 184) | 3.2 (1.2–5.2) [2.7–3.6] (n = 150) | .32 | 3.1 (1.6–4.1) [2.7–3.4] | 3.4 (1.4–5.2) [3.0–3.7] | <.001 |
| SGA | 30 (16) (n = 184) | 27 (18) (n = 149) | .66 | 19 (17) | 12 (14) | .55 |
| Apgar score at 5 min, median (range) [IQR] | 9 (5–10) [9–9] (n = 181) | 9 (3–10) [9–9] (n = 138) | .94 | 9 (5–10) [9–9] (n = 113) | 9 (3–10) [9–9] (n = 87) | .56 |
| Maternal characteristics | ||||||
| Maternal age at delivery, median (range) [IQR], y | 31.5 (16.7–45.3) [27.1–35.5] | 32.2 (16.7–44.0) [28.7–35.7] (n = 135) | .15 | 31.0 (17.4– 42.4) [26.8–35.2] | 32.5 (21.3– 43.0) [29.8–35.8] | .06 |
| Maternal ethnicity | ||||||
| Indigenous | 42 (13) | 9 (3) | <.001 | 33 (29) | 7 (8) | <.001 |
| African Caribbean black | 158 (49) | 2 (0.7) | 28 (25) | 2 (2) | ||
| White | 84 (26) | 93 (30) | 39 (34) | 55 (63) | ||
| Asian | 21 (6) | 29 (9) | 10 (9) | 16 (18) | ||
| Other/unknown | 12/7 (6) | 19/154 (57) | 4/0 (4) | 1/7 (9) | ||
| Maternal smoking ever in pregnancy | 105 (33) (n = 322) | 45 (30) (n = 152) | .51 | 63 (55) | 38 (43) | .09 |
| Detectable HIV pVL (>50 copies/mL) close to delivery | 18 (12) (n = 145) | NA | … | 12 (12) (n = 99) | NA | … |
| cART characteristics | ||||||
| Duration of in utero cART exposure, median (range) [IQR], wk | 24.4 (0.0– 41.7) [16.6–37.4] (n = 322) | NA | … | 20.6 (0.0–41.1) [14.8–36.6] | NA | … |
| Maternal cART initiation | ||||||
| Before conception | 124 (38) | NA | … | 40 (35) | NA | … |
| In 1st trimester | 39 (12) | NA | 15 (13) | NA | ||
| In 2nd trimester | 124 (38) | NA | 46 (40) | NA | ||
| In 3rd trimester | 29 (9) | NA | 13 (11) | NA | ||
| Naive/unknown | 7/1 (2) | NA | 0 (0) | NA | ||
| Maternal cART regimen | ||||||
| AZT + 3TC + NVP | 26 (8) | NA | … | 7 (6) | NA | … |
| AZT + 3TC + NFV | 83 (26) | NA | 34 (30) | NA | ||
| AZT + 3TC + LPV/r | 112 (35) | NA | 45 (39) | NA | ||
| ABC + 3TC + PI/r | 32 (10) | NA | 10 (9) | NA | ||
| TDF + FTC (or 3TC) + PI/r | 27 (8) | NA | 10 (9) | NA | ||
| Otherc | 36 (11) | NA | 8 (7) | NA | ||
| Naive/unknown | 7/1 (2) | NA | 0 (0) | NA | ||
| Infant AZT prophylaxis, median (range) [IQR], wk | 6 (0–8) [5–6] (n = 323)d | NA | … | 5 (2–6) [4–6] | NA | … |
Abbreviations: 3TC, lamivudine; ABC, abacavir; AZT, zidovudine; cART, combination antiretroviral therapy; CHEU, children who are HIV exposed and uninfected; CHUU, children who are HIV unexposed and uninfected; FTC, emtricitabine; HIV, human immunodeficiency virus; IQR, interquartile range; LPV/r, ritonavir-boosted lopinavir; NA, not applicable; NFV, nelfinavir; NVP, nevirapine; PI/r, ritonavir-boosted protease inhibitor; pVL, plasma viral load; SGA, small for gestational age; TDF, tenofovir disoproxil fumarate.
aTwenty-nine sets of siblings (25 CHEU and 4 CHUU) were identified among all study participants, of whom 5 were CHEU twin pairs. For birth analyses, only the first twin was included in the models. For the longitudinal model, all children were included. Three anonymous CHUU controls with a birth specimen but for whom no information apart from age and sex was available were not listed in the table.
bData represent no. (%) of children unless otherwise specified.
cThe “other” cART regimens are listed in Supplementary Table 2.
dAmong CHEU, 93 received AZT + 3TC postnatal prophylaxis.
Demographic and Clinical Characteristics of CHEU and CHUU, Including All Participants and Children With a Blood Specimen at Birtha
| Characteristics | All Participants, No. (%)b | Children With a Blood Specimen at Birth, No. (%)b | ||||
|---|---|---|---|---|---|---|
| CHEU (n = 324) | CHUU (n = 306) | P Value | CHEU (n = 114) | CHUU (n = 88) | P Value | |
| Infant characteristics | ||||||
| Male sex | 161 (50) | 169 (55) | .16 | 64 (56) | 47 (53) | .70 |
| Gestational age, median (range) [IQR], wk | 38.4 (27.1–41.7) [37.2–39.6] (n = 323) | 39.0 (28.3–42.1) [37.4–40.1] (n = 149) | .07 | 38.3 (31.3–41.6) [37.3–39.6] | 39.4 (28.9– 42.1) [38.2–40.3] | <.001 |
| Preterm delivery (<37 wk) | 61 (19) | 28 (19) | 23 (20) | 9 (10) | ||
| <37 and ≥34 wk | 42 (13) | 17 (11) | .98 | 19 (17) | 8 (9) | .055 |
| <34 wk | 19 (6) | 11 (7) | 4 (4) | 1 (1) | ||
| Birth weight, median (range) [IQR], kg | 3.1 (1.6–4.2) [2.7–3.4] (n = 184) | 3.2 (1.2–5.2) [2.7–3.6] (n = 150) | .32 | 3.1 (1.6–4.1) [2.7–3.4] | 3.4 (1.4–5.2) [3.0–3.7] | <.001 |
| SGA | 30 (16) (n = 184) | 27 (18) (n = 149) | .66 | 19 (17) | 12 (14) | .55 |
| Apgar score at 5 min, median (range) [IQR] | 9 (5–10) [9–9] (n = 181) | 9 (3–10) [9–9] (n = 138) | .94 | 9 (5–10) [9–9] (n = 113) | 9 (3–10) [9–9] (n = 87) | .56 |
| Maternal characteristics | ||||||
| Maternal age at delivery, median (range) [IQR], y | 31.5 (16.7–45.3) [27.1–35.5] | 32.2 (16.7–44.0) [28.7–35.7] (n = 135) | .15 | 31.0 (17.4– 42.4) [26.8–35.2] | 32.5 (21.3– 43.0) [29.8–35.8] | .06 |
| Maternal ethnicity | ||||||
| Indigenous | 42 (13) | 9 (3) | <.001 | 33 (29) | 7 (8) | <.001 |
| African Caribbean black | 158 (49) | 2 (0.7) | 28 (25) | 2 (2) | ||
| White | 84 (26) | 93 (30) | 39 (34) | 55 (63) | ||
| Asian | 21 (6) | 29 (9) | 10 (9) | 16 (18) | ||
| Other/unknown | 12/7 (6) | 19/154 (57) | 4/0 (4) | 1/7 (9) | ||
| Maternal smoking ever in pregnancy | 105 (33) (n = 322) | 45 (30) (n = 152) | .51 | 63 (55) | 38 (43) | .09 |
| Detectable HIV pVL (>50 copies/mL) close to delivery | 18 (12) (n = 145) | NA | … | 12 (12) (n = 99) | NA | … |
| cART characteristics | ||||||
| Duration of in utero cART exposure, median (range) [IQR], wk | 24.4 (0.0– 41.7) [16.6–37.4] (n = 322) | NA | … | 20.6 (0.0–41.1) [14.8–36.6] | NA | … |
| Maternal cART initiation | ||||||
| Before conception | 124 (38) | NA | … | 40 (35) | NA | … |
| In 1st trimester | 39 (12) | NA | 15 (13) | NA | ||
| In 2nd trimester | 124 (38) | NA | 46 (40) | NA | ||
| In 3rd trimester | 29 (9) | NA | 13 (11) | NA | ||
| Naive/unknown | 7/1 (2) | NA | 0 (0) | NA | ||
| Maternal cART regimen | ||||||
| AZT + 3TC + NVP | 26 (8) | NA | … | 7 (6) | NA | … |
| AZT + 3TC + NFV | 83 (26) | NA | 34 (30) | NA | ||
| AZT + 3TC + LPV/r | 112 (35) | NA | 45 (39) | NA | ||
| ABC + 3TC + PI/r | 32 (10) | NA | 10 (9) | NA | ||
| TDF + FTC (or 3TC) + PI/r | 27 (8) | NA | 10 (9) | NA | ||
| Otherc | 36 (11) | NA | 8 (7) | NA | ||
| Naive/unknown | 7/1 (2) | NA | 0 (0) | NA | ||
| Infant AZT prophylaxis, median (range) [IQR], wk | 6 (0–8) [5–6] (n = 323)d | NA | … | 5 (2–6) [4–6] | NA | … |
| Characteristics | All Participants, No. (%)b | Children With a Blood Specimen at Birth, No. (%)b | ||||
|---|---|---|---|---|---|---|
| CHEU (n = 324) | CHUU (n = 306) | P Value | CHEU (n = 114) | CHUU (n = 88) | P Value | |
| Infant characteristics | ||||||
| Male sex | 161 (50) | 169 (55) | .16 | 64 (56) | 47 (53) | .70 |
| Gestational age, median (range) [IQR], wk | 38.4 (27.1–41.7) [37.2–39.6] (n = 323) | 39.0 (28.3–42.1) [37.4–40.1] (n = 149) | .07 | 38.3 (31.3–41.6) [37.3–39.6] | 39.4 (28.9– 42.1) [38.2–40.3] | <.001 |
| Preterm delivery (<37 wk) | 61 (19) | 28 (19) | 23 (20) | 9 (10) | ||
| <37 and ≥34 wk | 42 (13) | 17 (11) | .98 | 19 (17) | 8 (9) | .055 |
| <34 wk | 19 (6) | 11 (7) | 4 (4) | 1 (1) | ||
| Birth weight, median (range) [IQR], kg | 3.1 (1.6–4.2) [2.7–3.4] (n = 184) | 3.2 (1.2–5.2) [2.7–3.6] (n = 150) | .32 | 3.1 (1.6–4.1) [2.7–3.4] | 3.4 (1.4–5.2) [3.0–3.7] | <.001 |
| SGA | 30 (16) (n = 184) | 27 (18) (n = 149) | .66 | 19 (17) | 12 (14) | .55 |
| Apgar score at 5 min, median (range) [IQR] | 9 (5–10) [9–9] (n = 181) | 9 (3–10) [9–9] (n = 138) | .94 | 9 (5–10) [9–9] (n = 113) | 9 (3–10) [9–9] (n = 87) | .56 |
| Maternal characteristics | ||||||
| Maternal age at delivery, median (range) [IQR], y | 31.5 (16.7–45.3) [27.1–35.5] | 32.2 (16.7–44.0) [28.7–35.7] (n = 135) | .15 | 31.0 (17.4– 42.4) [26.8–35.2] | 32.5 (21.3– 43.0) [29.8–35.8] | .06 |
| Maternal ethnicity | ||||||
| Indigenous | 42 (13) | 9 (3) | <.001 | 33 (29) | 7 (8) | <.001 |
| African Caribbean black | 158 (49) | 2 (0.7) | 28 (25) | 2 (2) | ||
| White | 84 (26) | 93 (30) | 39 (34) | 55 (63) | ||
| Asian | 21 (6) | 29 (9) | 10 (9) | 16 (18) | ||
| Other/unknown | 12/7 (6) | 19/154 (57) | 4/0 (4) | 1/7 (9) | ||
| Maternal smoking ever in pregnancy | 105 (33) (n = 322) | 45 (30) (n = 152) | .51 | 63 (55) | 38 (43) | .09 |
| Detectable HIV pVL (>50 copies/mL) close to delivery | 18 (12) (n = 145) | NA | … | 12 (12) (n = 99) | NA | … |
| cART characteristics | ||||||
| Duration of in utero cART exposure, median (range) [IQR], wk | 24.4 (0.0– 41.7) [16.6–37.4] (n = 322) | NA | … | 20.6 (0.0–41.1) [14.8–36.6] | NA | … |
| Maternal cART initiation | ||||||
| Before conception | 124 (38) | NA | … | 40 (35) | NA | … |
| In 1st trimester | 39 (12) | NA | 15 (13) | NA | ||
| In 2nd trimester | 124 (38) | NA | 46 (40) | NA | ||
| In 3rd trimester | 29 (9) | NA | 13 (11) | NA | ||
| Naive/unknown | 7/1 (2) | NA | 0 (0) | NA | ||
| Maternal cART regimen | ||||||
| AZT + 3TC + NVP | 26 (8) | NA | … | 7 (6) | NA | … |
| AZT + 3TC + NFV | 83 (26) | NA | 34 (30) | NA | ||
| AZT + 3TC + LPV/r | 112 (35) | NA | 45 (39) | NA | ||
| ABC + 3TC + PI/r | 32 (10) | NA | 10 (9) | NA | ||
| TDF + FTC (or 3TC) + PI/r | 27 (8) | NA | 10 (9) | NA | ||
| Otherc | 36 (11) | NA | 8 (7) | NA | ||
| Naive/unknown | 7/1 (2) | NA | 0 (0) | NA | ||
| Infant AZT prophylaxis, median (range) [IQR], wk | 6 (0–8) [5–6] (n = 323)d | NA | … | 5 (2–6) [4–6] | NA | … |
Abbreviations: 3TC, lamivudine; ABC, abacavir; AZT, zidovudine; cART, combination antiretroviral therapy; CHEU, children who are HIV exposed and uninfected; CHUU, children who are HIV unexposed and uninfected; FTC, emtricitabine; HIV, human immunodeficiency virus; IQR, interquartile range; LPV/r, ritonavir-boosted lopinavir; NA, not applicable; NFV, nelfinavir; NVP, nevirapine; PI/r, ritonavir-boosted protease inhibitor; pVL, plasma viral load; SGA, small for gestational age; TDF, tenofovir disoproxil fumarate.
aTwenty-nine sets of siblings (25 CHEU and 4 CHUU) were identified among all study participants, of whom 5 were CHEU twin pairs. For birth analyses, only the first twin was included in the models. For the longitudinal model, all children were included. Three anonymous CHUU controls with a birth specimen but for whom no information apart from age and sex was available were not listed in the table.
bData represent no. (%) of children unless otherwise specified.
cThe “other” cART regimens are listed in Supplementary Table 2.
dAmong CHEU, 93 received AZT + 3TC postnatal prophylaxis.
Seven (2%) CHEU were not exposed to cART in utero and were excluded from analyses at birth. Among the remaining 317 CHEU, cART exposure started at conception for 124 (38%), and during the first, second, and third trimesters for 39 (12%), 124 (38%), and 29 (9%), respectively. The median (interquartile range) duration of in utero cART exposure was 24.4 (16.6–37.4) weeks. The majority of CHEU were exposed to a combination of zidovudine (AZT) plus lamivudine (3TC) (n = 221 [69%]), with either ritonavir-boosted lopinavir (n = 112 [35%]), nelfinavir (n = 83 [36%]), or nevirapine (n = 26 [8%]) (for details see Table 1 and Supplementary Table 2). All CHEU received AZT prophylaxis for a median (interquartile rand) of 6 (5–6) weeks.
Participants With a Birth Specimen
Demographic characteristics for the 114 CHEU and 88 CHUU with a birth specimen (obtained 0–3 days after birth) are presented in Table 1. In addition to the imbalance in ethnicity whereby mothers of CHEU were more often Indigenous (29%) or African Caribbean black (25%) compared with CHUU (8% and 2%, respectively), CHEU had lower gestational age and weight at birth (both P < .001). All other characteristics were similar between groups, although rates of maternal smoking during pregnancy were higher (55% in the CHEU and 43% in the CHUU group) than the corresponding all-participant groups (Table 1).
Infant mtDNA Content at Birth
MtDNA content at birth was analyzed in 114 CHEU and 86 CHUU, as measurements for 2 CHUU failed assay quality control. In univariate analyses, CHEU had significantly higher mtDNA content at birth than CHUU (Figure 1A). Lower gestational age (Supplementary Figure 2 and Supplementary Table 3) and prematurity (Figure 1B and Supplementary Table 3) were significantly associated with higher mtDNA content at birth in both CHEU and CHUU. Maternal smoking during pregnancy was also associated with higher mtDNA content at birth overall, but this was primarily influenced by the CHUU group (Figure 1C and Supplementary Table 3). A significant interaction between CHEU and CHUU status and maternal smoking (P = .04) was detected and was later included in the multivariable models. Univariately, CHEU exposed to a combination of AZT, 3TC, and ritonavir-boosted lopinavir (P = .02) or abacavir, 3TC, and ritonavir-boosted protease inhibitor (PI) (P = .02) in utero had significantly higher mtDNA content at birth compared with CHUU controls, although some groups were small (Figure 1D). Finally, infant sex and ethnicity were not associated with mtDNA content at birth (Supplementary Table 3, and Supplementary Figure 3).
Unadjusted comparisons of mitochondrial DNA (mtDNA) content at birth. A, children who are HIV exposed and uninfected (CHEU) and children who are HIV unexposed and uninfected (CHUU) (Student t test). B, Infants born at term or preterm (Mann-Whitney U test). C, CHEU and CHUU exposed or not exposed to maternal smoking during pregnancy (Student t test). D, CHEU exposed to different combination antiretroviral therapy (cART) regimens in utero and CHUU. P values were determined with Kruskal-Wallis test (dashed brackets) or Dunn’s test (adjusted for multiple comparisons) (solid brackets); for all panels, whiskers of the box plots represent the 5th–95th percentiles. Abbreviations: 3TC, lamivudine; ABC, abacavir; AZT, zidovudine; FTC, emtricitabine; LPV/r, ritonavir-boosted lopinavir; NFV, nelfinavir; NVP, nevirapine; PI/r, ritonavir-boosted protease inhibitor; TDF, tenofovir disoproxil fumarate.
Within the CHEU group, duration of in utero cART exposure was not associated with mtDNA content at birth, when analyzed either as weeks of exposure or trimester of maternal cART initiation (Supplementary Table 3). In addition, there were no differences in CHEU mtDNA content at birth between cART types.
Strong collinearity precluded the inclusion of gestational age and birth weight in the same model. While the 2 models were similar, gestational age yielded a better fit. In the final multivariable model of all participants (n = 192) Figure 2A) that included CHEU/CHUU status, gestational age at birth, maternal smoking, and the CHEU/CHUU status * maternal smoking interaction term, lower gestational age at birth was independently associated with higher mtDNA content at birth. This model further suggests that CHEU status and being born to mothers who smoked were also associated with higher mtDNA content. However, this must be interpreted with caution, given the behavior of the interaction term, which, together with the CHEU and CHUU models (Figure 2C and 2D), clearly implies that the association between smoking and mtDNA is restricted to CHUU who show high mtDNA content when born to smoking mothers (Figure 1C).
Multivariable regression analyses of the association between possible predictors and infant mitochondrial DNA (mtDNA) content at birth. A, All participants (n = 192; R2 = 0.14). B, All term infants only (n = 161; R2 = 0.12). C, Children who are HIV exposed and uninfected (CHEU) (n = 106; R2 = 0.05). D, Children who are HIV unexposed and uninfected (CHUU) (n = 86, R2 = 0.18). Effect sizes are expressed as nonstandardized β values. Note that 8 CHEU who were exposed to a nonstandard combination antiretroviral therapy regimen in utero were excluded from these analyses. Abbreviations: CI, confidence interval; GA, gestational age.
Given the strong relationship between prematurity and higher mtDNA content at birth, and because few CHUU were preterm, a sensitivity analysis that included only children born at term (n = 161; 84 CHEU and 77 CHUU) was performed. The results were similar to those in the full sample, with a weakened association with gestational age (Figure 2B).
In light of the imbalance in ethnicity between the 2 study groups, a second sensitivity analysis that included only children born to white mothers (n = 93; 39 CHEU and 54 CHUU) was performed. Lower gestational age at birth remained independently associated with higher mtDNA content at birth, while the effect of HIV/cART exposure was weakened (Supplementary Figure 4).
Finally, to investigate the potential relationship between infant mtDNA and maternal mtDNA, we performed a sensitivity analysis in a subset of children (n = 81; 47 CHEU and 34 CHUU) for whom maternal WB specimens collected close to delivery (31–38 weeks of gestation) were available. Maternal mtDNA was similar in both groups and weakly correlated with infant birth mtDNA (Figure 3A). The multivariable model showed that higher maternal mtDNA was independently associated with higher infant mtDNA but that CHEU status and lower gestational age also remained independently associated (Figure 3B).
A, Pearson correlation showing the relationship between infant mitochondrial DNA (mtDNA) content at birth and maternal mtDNA content close to delivery (31–38 weeks gestation). B, Multivariable regression analysis among participants for whom maternal mtDNA content close to delivery was available (sensitivity analysis of infant and mother pairs) showing the association between possible predictors and infant mtDNA content at birth (n = 81; R2 = 0.24). Effect sizes are expressed as nonstandardized β values. Of note, the results were unchanged when maternal mtDNA content was excluded from the model for these 81 participants. Abbreviations: CHEU, children who are human immunodeficiency virus (HIV) exposed and uninfected; CHUU, children who are HIV unexposed and uninfected; CI, confidence interval; GA, gestational age.
In secondary models (Supplementary Figure 5), we explored whether the type of in utero cART exposure was associated with infant mtDNA at birth and detected no association (Supplementary Figure 5A). However, among the subset of 161 infants born at term (Supplementary Figure 5B), we observed a modest association between in utero exposure to boosted PI–based cART and higher mtDNA content at birth; the effect sizes were similar for nonbooster PI-based and nonnucleoside reverse-transcriptase inhibitor–based cART, but the 95% confidence interval were wider.
Cross-Sectional Comparison of mtDNA Content During the First 3 Years of Life
For this analysis, 214 distinct CHEU were age and sex matched 1:1 with 214 CHUU (Supplementary Table 4). The 2 groups showed nearly identical slopes for the linear regressions of mtDNA content versus age. However, the intercepts (elevation) indicated that CHEU have significantly higher mtDNA content on average than CHUU throughout the first 3 years of life (P < .001) (Figure 4 and Supplementary Figure 6). The results were unchanged in sensitivity analyses excluding children pairs containing either a preterm child (Supplementary Figure 7A) or a child exposed to maternal smoking (Supplementary Figure 7B).
Relationship between mitochondrial DNA (mtDNA) content and age during the first 3 years of life among age- and sex-matched children. CHEU, children who are human immunodeficiency virus (HIV) exposed and uninfected; CHUU, children who are HIV unexposed and uninfected.
Longitudinal mtDNA Content Dynamics Among CHEU
Longitudinal changes in mtDNA content over the first 3 years of life were assessed for the 214 CHEU for whom ≥ 2 blood specimens were available. A significant nonlinear relationship was observed, with mtDNA content remaining relatively unchanged during the first 6 months of life, followed by a gradual decrease to age 2 years and a leveling thereafter (Figure 5A). The fit of this additive mixed-effects model was only marginally better than that of a linear mixed-effects model (likelihood ratio test, P = .03) that suggested a gradual decrease of 20 mtDNA copies per year (P < .001) (Supplementary Figure 8), a rate similar to that seen for the CHUU group’s linear regression (single specimen per participant; P < .001) (Supplementary Figure 8).
A, Nonlinear regression model (generalized mixed effects additive model) of mitochondrial DNA (mtDNA) content and age (in months) for children who are human immunodeficiency virus exposed and uninfected (CHEU). B, Comparison of mtDNA content at birth (aged 0–3 days) and the subsequent visit (age 18–47 days) among CHEU (paired Student t test).
CHEU mtDNA Content During Prophylaxis
We used paired CHEU specimens spanning the 6-week postnatal AZT prophylaxis period and detected no difference in mtDNA content between birth and the closest subsequent visit during prophylaxis, which was at a median (range) age of 31 (18–47) days (n = 58; P = .11) (Figure 5B).
DISCUSSION
Consistent with previous studies by our group [32, 35] and others [33, 34], we observed an association between HIV/cART exposure in utero and elevated blood mtDNA content at birth compared with CHUU controls, an effect that persisted throughout the first 3 years of life. Incidentally, CHEU exposed to ritonavir-boosted PI regimens had modestly higher mtDNA content at birth compared with CHUU; a similar trend was observed with exposure to nonboosted PI, but we lacked power to compare nucleoside reverse-transcriptase inhibitors–based regimens with PI-based regimens in this study. It is noteworthy that a majority of the participants in 2 other US studies reporting higher CHEU mtDNA content were also exposed to PI-containing maternal regimens [33, 34]. Although the mechanism of this effect is unclear, in vitro studies have shown that PIs (boosted and nonboosted) increase reactive oxygen species and can cause oxidative stress, disrupt mitochondrial intermembrane potential, and increase cellular apoptosis [39]. All these may plausibly result in altered mtDNA levels. Potential mechanisms include adaptive mitochondrial biogenesis, altered proportions of immune cell subsets with different mtDNA content, or a dysregulation of mitochondrial replication/mitophagy homeostasis in response to in utero HIV and/or cART-associated stresses. Our study was not designed to address putative mechanism(s).
Our findings are in contrast with those of older studies that reported lower mtDNA levels in CHEU [27–31]. However, CHEU exposed to PIs were scarce in these studies, with most pregnant women receiving either single or dual AZT-containing regimens. Others were exposed to stavudine or didanosine, both known to decrease mtDNA [21, 40–42]. Most of these studies acknowledge limitations related to sample size and/or heterogeneity in the recruitment of study and control participants, a possible source of bias. In addition, heterogeneity in the type of specimens studied may partially explain discrepancies between studies.
We observed an independent association between lower gestational age and higher blood mtDNA content at birth; however, this effect was primarily driven by preterm children born before 37 weeks of gestation. Another group reported a similar negative correlation between umbilical cord blood mtDNA content and gestational age at birth [43]. A recent study reported higher cord blood mtDNA content in the context of placental insufficiency characterized by preeclampsia and/or intrauterine growth restriction, but not in association with preterm birth per se. However, their study sample contained only 8 preterm births, selected for their lack of preeclampsia and/or intrauterine growth restriction, which may have limited power and/or induced bias [44]. We are not aware of any other published study investigating infant mtDNA in preterm birth. It is possible that mtDNA content naturally declines toward the end of the gestational period, and this could explain our observation of higher mtDNA content among preterm children. However, given the association of higher mtDNA with smoking, it could be linked to a stress response related to being born preterm. Studies in animal models or larger prospective cohorts of blood and other tissues would be required to investigate this.
Of note, CHEU have a >2-fold higher risk of being born preterm compared with CHUU [2, 45–47], something we observed in our sample among children with a birth specimen. However, the similar preterm birth rates observed within the full sample, approximately 20% in both groups, suggests an apparent overrepresentation of preterm children in our CHUU group. This may be related to biases associated with participant recruitment. HIV-uninfected controls were a convenience sample recruited at the hospital and clinics during routine visits to their pediatricians; it is possible that children born preterm would have more frequent visits, favoring their recruitment. Alternatively, as we made a deliberate effort to enroll control children with similar sociodemographic characteristics as the CHEU group, CHUU may have had an inherently higher preterm birth risk compared with the general population.
Within the CHUU group, our finding of higher mtDNA content at birth in association with maternal smoking during pregnancy is consistent with another report of cord serum findings in children exposed to tobacco smoke prenatally [48]. This may be related to adaptive mitochondrial biogenesis in response to smoking-induced oxidative stress, an effect possibly masked by HIV/cART exposure among CHEU.
Although not the focus of our study, a modest relationship between infant and maternal mtDNA was observed. It is possible that alterations in infant mtDNA levels reflect the mother’s mtDNA levels, which may in turn be influenced by maternal HIV/cART or other in utero stresses derived from it. Future studies investigating the longitudinal trajectory of maternal mtDNA during pregnancy may help explain the relationship between the two.
Finally, our longitudinal analysis revealed that CHEU mtDNA levels remain relatively unchanged very early in life and gradually decrease thereafter. The implications of higher mtDNA content at birth and in early life remains unclear, given that both lower and higher mtDNA content have been associated with cancer and other diseases, fertility, organ development, and aging in the general population [15]. Further investigations are required to determine the clinical meaning of harboring higher mtDNA content and whether this is associated with health outcomes later in life.
Ours is the largest longitudinal study of mtDNA content in CHEU during the first 3 years of life. However, we lacked longitudinal specimens from CHUU, and this precluded us from conducting detailed comparisons between groups. Platelets contain mtDNA, and information on platelet count was unavailable. However, disorders related to low platelet count, such as thrombocytopenia may be exacerbated, by HIV/cART exposure, and would present a conservative bias. Furthermore, we observed no correlation between platelet count and mtDNA content in a previous study of older CHEU [35].
Our cohort had some demographic and behavioral imbalances. Nearly half the CHEU but <1% of CHUU were African Caribbean black. However, we did not observe any noticeable differences in CHEU and CHUU mtDNA content at birth based on ethnicity. Furthermore, when forced into the multivariable models, ethnicity increased the Akaike information criteria and decreased the overall fit of the model (data not shown). Together with our sensitivity analysis restricted to white children, these data suggest that ethnicity is not an important predictor of mtDNA content at birth. Rates of maternal smoking during pregnancy were high for both CHEU and CHUU. Given that the association between maternal smoking during pregnancy and higher mtDNA content at birth was observed only among CHUU, this may be related to differences in smoking behavior (frequency, intensity, and duration) between groups.
Based on our previous study [36], the majority of women who reported smoking at any time during their pregnancy continued doing so throughout, with very few quitting. Finally, we lacked information on maternal HIV load, CD4 count, comorbid conditions, coinfections including hepatitis C virus and cytomegalovirus, illicit substance use, mental health, and other maternal exposures that may have confounded our results.
In conclusion, higher mtDNA content at birth was associated with exposures to stressors, such as maternal HIV and smoking, or being born preterm, itself possibly related to stresses. This may represent a rebound following removal of said stressor(s) when the child is born. These results merit further investigations on the long-term mitochondrial effects of HIV/cART exposure and the potential implications of altered mtDNA content as a predictor of health in this expanding population of HIV-uninfected, cART-exposed individuals.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Notes
Acknowledgments. We thank all the study participants and the research staff at our Children and Women: AntiRetrovirals and Markers of Aging (CARMA) sites and in the Côté laboratory.
Other investigators in the CIHR Team in Cellular Aging and HIV Comorbidities in Women and Children (CARMA) include Neora Pick, MD, Melanie Murray, MD, Patricia Janssen, PhD, Joel Singer, PhD, Normand Lapointe, MD, Jerilynn Prior, MD, Michael Silverman, MD, and Mary Lou Smith, PhD.
Author contributions. Study design: A. Ajaykumar, F. K., J. B., A. B., A. Alimenti, H. S., D. M. M., and H. C. F. C. Clinical data collection: F. K., J. B., A. B., A. Alimenti, H. S., and D. M. M. Conducting of assays: A. Ajaykumar., M. Z., and S. S. Data analysis: A. Ajaykumar. Longitudinal data analyses: A. Y. K. A. Overseeing of laboratory data collection: H. C. F. C. Preparation of figures: A. Ajaykumar., and A. Y. K. A. Writing first draft of manuscript: A. Ajaykumar. Reading and editing of manuscript: M. Z., F. K., J. B., A. B., A. Alimenti, H. S., S. S., A. Y. K. A., D. M. M., and H. C. F.C.
Financial support. This research was supported in part by the Canadian Foundation for AIDS Research (grants 20-004 and 16-012), the Canadian Institutes of Health Research (team grants HET-85515 [HIV Therapy and Aging; 2007–2012] and TCO-125269 [Cellular Aging and HIV Comorbidities in Women and Children; 2013–2018] to D. M. M. and H. C. F. C.), the University of British Columbia Centre for Blood Research (internal collaborative training award to A. Ajaykumar), the University of British Columbia Department of Pathology and Laboratory Medicine (summer studentship award to M. Z.), and Fonds de la Recherche du Québec-Santé (Junior 1 career scholarship to F. K.).
Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
Presented in part: 25th Annual Conference on Retroviruses and Opportunistic Infections, Boston, Massachusetts, 4–7 March 2018; and 27th Annual Canadian Conference on HIV/AIDS Research, Vancouver, British Columbia, Canada, 26–29 April 2018.
References
Author notes
See Acknowledgments for list of other investigators on the CIHR Team in Cellular Aging and HIV Comorbidities in Women and Children (Children and Women: AntiRetrovirals and Markers of Aging [CARMA]).
