Influence of Exposure to ART and HIV Infection on TL in HIV-Infected Mothers and Their HEU Children Compared With Uninfected, Unexposed Healthy Controls (PR/CARMA Cohort)
HEU infants born to HIV-infected, ART-treated mothers and uninfected children born to HIV-uninfected control mothers were similar in terms of gestational age and weight at birth, gender, Apgar score at 5 minutes, delivery method, and maternal age, although there were differences with respect to ethnicity. The HIV-infected, ART-exposed group included more aboriginal people and fewer Asians than the control group and no black or Hispanic women (Table 2). A history of HCV infection was more common in HIV-infected, ART-exposed mothers than in controls. As data on HCV infection was not available for many control mothers (because it is not part of routine screening in HIV-negative pregnancies), it was not included in the statistical model. Of note, when available, HCV infection was highly correlated with the use of drugs of addiction. Smoking and use of drugs of addiction and/or methadone ever during pregnancy were well balanced between the 2 groups. All the mothers who reported smoking marijuana during pregnancy also smoked cigarettes, and of the mothers who received methadone during pregnancy, 81% (n = 21 of 26 in both groups) also used drugs of addiction. In the PR/CARMA cohort, 11 (19%) HIV-infected women conceived while on ART and remained on treatment, whereas 9 (15%) started ART in the first, 33 (56%) in the second, and 6 (10%) in the third trimester, for a median in utero ART exposure of 19.3 weeks, which was not statistically different in the SJ cohort (P = 0.66). The majority of the women in the PR/CARMA cohort were treated with AZT + 3TC + protease inhibitor (±ritonavir) (n = 41, 69%) and the women who conceived on ART were more likely to be on alternate regimens (n = 6, 55%). Half (30 of 59) of the HIV-infected women in this cohort were ART exposed before the studied pregnancy. Near delivery, median CD4+ T-cell count was 450 cells per microliter and 17% of women had a detectable pVL, with a median of 321 HIV RNA copies per milliliter plasma.
Peripheral blood leukocyte TL was compared between mothers and infants within and between the HIV-infected ART-exposed group and the control group. Again, maternal TL was significantly shorter than that of their infants (P < 0.001). However, no statistically significant TL differences were observed between HIV-infected and control mothers, or between their infants, before or after adjusting for covariates (Table 3; Fig. 1B). In the PR/CARMA cohort, infant TL correlated with maternal TL (Fig. 2) and more strongly correlated with cord blood and placenta TL than maternal TL was, whether considering all samples or each group separately (for detailed correlations between samples, see Table 3, Supplemental Digital Content, http://links.lww.com/QAI/A329).
Cord blood leukocyte TL, as measured by quantitative PCR, was significantly shorter in the HIV-infected, ART-exposed group than in the control group (P = 0.04). After controlling for maternal age, maternal ethnicity, gestational age at delivery, smoking ever, and use of drugs of addiction/methadone ever in pregnancy, this difference became more marginally significant (P = 0.06). None of the covariates examined were independently associated with TL in peripheral or cord blood.
In placental tissue, TL strongly correlated between the maternal and foetal sides of the organ in both groups (see Table 3, Supplemental Digital Content http://links.lww.com/QAI/A329), and there were no statistically significant relationships between gestational age at delivery or maternal age and TL. No differences in TL were observed between the 2 groups in unadjusted comparisons (Table 3). However, after adjusting for covariates, placenta from HIV-infected, ART-exposed pregnancies showed shorter TL on the maternal side than controls (P = 0.032) and a similar trend was noted in samples obtained from the foetal side of the organ (P = 0.08). Smoking in pregnancy was independently associated with shorter placental TL on the maternal side (P = 0.041) and tended similarly on the foetal side (P = 0.055).
Among HIV-infected, ART-exposed subjects from the PR/CARMA cohort, the following variables were also considered as potential predictors of TL in linear regression models: duration of maternal HIV infection, duration of maternal prepregnancy ART exposure, length of infant in utero ART exposure, and pVL and CD4+ T-cell counts near delivery. None of these variables was associated with infant TL or placental TL on the maternal side. However, higher CD4+ T-cell counts (P = 0.047) and longer duration of HIV infection at delivery (P = 0.016) were associated with shorter maternal TL (n = 52). In similar regression models, higher CD4+ T-cell counts (P = 0.018), higher pVL (P = 0.009), and older maternal age (P = 0.043) were associated with longer cord blood TL (n = 47). Finally, higher HIV pVL was associated with longer placental TL on the foetal side (P = 0.028).
Exposure to ART or HIV/ART and TL
Results obtained in our retrospective cohort study (SJ cohort) comparing TL in HIV-infected mothers treated or untreated with ART in pregnancy and in their HEU infants did not reveal significantly different TLs in maternal or infant peripheral blood leukocytes. This suggests that in the context of pregnancy, perinatal exposure to ART itself is not associated with shorter TL in infants, a reassuring finding. Further comparison of HIV-infected ART-treated mother/infant pairs, this time with uninfected control mothers and their infants as part of the prospective PR/CARMA cohort study, also did not detect any significant TL difference between the groups, suggesting that maternal HIV infection does not exert an important effect on TL. In the PR/CARMA cohort, maternal TL positively correlated with both infant blood and cord blood TL, in accordance with other studies.16,17 Some differences emerged in cord blood and placenta, whereby shorter TL were seen in the HIV/ART-exposed group compared with uninfected controls. Interestingly, the mean TL value for each of the 7 sample types investigated within the 2 cohorts was consistently lower in the exposed group than in the unexposed one (Table 3). Although the difference in TL reached significance in a few instances only, such unidirectional change in TL between the groups is unlikely to be coincidental. It is noteworthy that the greatest decrease in TL was seen in cord blood, a tissue that contains telomerase-expressing hematopoietic, mesenchymal, and endothelial stem cells in higher abundance than peripheral blood.18 As hematopoietic stem cells are the precursors of other cord blood nucleated cells, inhibition of telomerase activity by ART could exert the most impact on TL within this compartment. The larger effect seen in cord blood compared with infant peripheral blood could also reflect its distinct rather than linear origin, as recently suggested.19 Nevertheless, this observation raises concern on the potential effect of NRTI on stem cells and requires further study.
Importantly, although ART exposure itself did not emerge as an important predictor of shorter TL in mothers or infants, some well-recognized health risk factors did. Smoking and use of drugs of addiction ever in pregnancy was associated with shorter TL in mothers and in their infants in the SJ cohort. Both are known sources of oxidative stress,20–22 a major cause of telomere attrition through decreased TERT expression, which is regulated by redox-sensitive transcription factors,23–26 and/or telomeric DNA oxidative damage.23 In a mouse model, cigarette smoke exposure was shown to induce oxidative stress, telomere shortening, and apoptosis.27 In tissues that have higher expression of telomerase, namely, cord blood and placenta, the association observed between higher pVL and longer placenta TL among HIV/ART-treated PR/CARMA subjects could suggest some placental telomerase inhibition in the presence of effective ART. However, although foetal growth retardation has previously been associated with reduced placental telomerase activity28 and accelerated placenta telomere shortening,29 we did not observe any difference in infant birth weight in the ART-exposed groups compared with both the untreated (SJ cohort) or uninfected groups (PR/CARMA cohort). Among HIV+/ART-treated mothers in the PR/CARMA cohort, duration of HIV infection, which may be a surrogate indicator of exposure to inflammation and oxidative stress, was also associated with shorter TL. Taken together, our results suggest that the negative effects of known risk factors, such as smoking, use of drugs of addiction, and inflammation, likely exert greater influence than ART on the leukocyte TL of mothers and their infants.
Age and TL
As expected,30,31 mothers had shorter TL than their infants in both cohorts. Furthermore, in agreement with previous studies, TL was longest in placental tissue,32 followed by cord blood, infant, and maternal peripheral blood leukocytes.33 In both cohorts, maternal and infant TL positively, albeit weakly, correlated, but maternal age and maternal TL showed no correlation, likely because the age of the mothers (interquartile range = 26–35 years) spans a time of high interindividual variability and relative leveling of telomere attrition rate compared with rates seen at younger or older ages.34 In regression analyses, older maternal age was associated with longer TL in both the infants' peripheral blood and cord blood. This seemingly counterintuitive observation is in agreement with previous cohort studies that demonstrated a clear relationship between older parental age and longer TL in progeny,35,36 presumably a result of telomerase activity in germ line cells.36 Although this relationship is strongest with paternal age, it has also been reported on the maternal side in a large cohort.17 Paternal age or paternal ethnicity data were not available for this study, but as there was no difference in the maternal ages between the groups, it is plausible that there would be little difference in paternal ages.
Other limitations of this study include the higher interindividual coefficients of variation we observed for control infant peripheral blood leukocyte TL and cord blood TL (both 20%–25%), compared with those reported in the literature16,37 when this study was designed. This challenged the power of our study to detect small differences in TL. However, the cord blood TL variability we observed is in agreement with a more recent study, published after we completed data collection.38 Given that in the prospective observational cohort, there were no women who were HIV infected and not treated with ART, it was not possible to ascertain whether any TL differences observed were related to HIV, ART, or both. Finally, peripheral blood leukocyte TLs were generally longer in the SJ cohort than in the PR/CARMA cohort, and some of the associations with TL were not reproduced in both cohorts. Potential explanations include, but are not restricted to, the fact that different samples were collected (dried blood spot vs. whole blood) and stored for different lengths of time. The ethnic makeup of the 2 cohorts also differed considerably, and the threshold for categorizing a subject as smoking/using drugs of addiction may have differed slightly between the 2 cohorts.
In conclusion, our results on TL in mother and infant peripheral blood suggest that oxidative stress related to smoking or use of drugs of addiction is a stronger factor affecting TL than HIV and/or ART exposure. Advocating a healthy lifestyle for HIV-infected women could play an important role in counteracting any negative effect of HIV or ART on their child's future health. In tissues that express higher telomerase activity, such as cord blood and placenta, reduced exposure to ART, as reflected by higher pVL, may better preserve telomeres. Maternal HIV infection itself also seems to have minimal effect on infant TL, although in HIV-infected mothers, longer duration of HIV infection, which could reflect a greater exposure to inflammation and oxidative stress, may explain the association seen with shorter maternal TL.
At a time when programs involving prevention of mother-to-child HIV transmission with ART are being extended to large numbers of mothers and infants in the developed and developing countries, our observations suggesting that ART does not significantly affect TL in the peripheral blood of ART-exposed mothers and their infants are reassuring. However, the trend observed toward shorter TL in exposed samples in general, and in cord blood in particular, warrants further study. This is especially true considering several reports of possible long-term side effects associated with ART exposure.39–42 These studies at the cellular level represent an important complement to long-term clinical studies on ART in the context of pregnancy.
The authors acknowledge Laura Oliveira, Silvie Valois, Martine Caty, Johanne Samson, Tessa Chaworth-Musters, Daljeet Mahal, Judy Wong, Katherine Heath, and Michael Papsdorf for their contribution to this study, and Fatima Kakkar for critical reading of the manuscript.
1. Panel on Treatment of HIV-Infected Pregnant Women and Prevention of Perinatal Transmission. Recommendations for Use of Antiretroviral Drugs in Pregnant HIV-1-Infected Women for Maternal Health and Interventions to Reduce Perinatal HIV Transmission in the United States. 2011. 1–207. Available at: http://aidsinfo.nih.gov/contentfiles/PerinatalGL.pdf
. Accessed October 30, 2011.
2. World Health Organization. Antiretroviral Drugs for Treating Pregnant Women and Preventing HIV Infection in Infants. Geneva, Switzerland: World Health Organization; 2010.
3. Chappuy H, Treluyer JM, Jullien V, et al.. Maternal-fetal transfer and amniotic fluid accumulation of nucleoside analogue reverse transcriptase inhibitors in human immunodeficiency virus-infected pregnant women. Antimicrob Agents Chemother. 2004;48:4332–4336.
4. Heidari S, Mofenson L, Cotton MF, et al.. Antiretroviral drugs for preventing mother-to-child transmission of HIV: a review of potential effects on HIV-exposed but uninfected children. J Acquir Immune Defic Syndr. 2011;57:290–296.
5. Peng Y, Mian IS, Lue NF. Analysis of telomerase processivity: mechanistic similarity to HIV-1 reverse transcriptase and role in telomere maintenance. Mol Cell. 2001;7:1201–1211.
6. Gillis AJ, Schuller AP, Skordalakes E. Structure of the Tribolium castaneum
telomerase catalytic subunit TERT. Nature. 2008;455:633–637.
7. Gomez DE, Tejera AM, Olivero OA. Irreversible telomere shortening by 3'-azido-2',3'-dideoxythymidine (AZT) treatment. Biochem Biophys Res Commun. 1998;246:107–110.
8. Olivero OA. Relevance of experimental models for investigation of genotoxicity induced by antiretroviral therapy during human pregnancy. Mutat Res. 2008;658:184–190.
9. Datta A, Bellon M, Sinha-Datta U, et al.. Persistent inhibition of telomerase reprograms adult T-cell leukemia to p53-dependent senescence. Blood. 2006;108:1021–1029.
10. Olivero OA, Anderson LM, Diwan BA, et al.. Transplacental effects of 3'-azido-2',3'-dideoxythymidine (AZT): tumorigenicity in mice and genotoxicity in mice and monkeys. J Natl Cancer Inst. 1997;89:1602–1608.
11. Olivero OA, Fernandez JJ, Antiochos BB, et al.. Transplacental genotoxicity of combined antiretroviral nucleoside analogue therapy in Erythrocebus patas
monkeys. J Acquir Immune Defic Syndr. 2002;29:323–329.
12. Saliques S, Zeller M, Lorin J, et al.. Telomere length and cardiovascular disease. Arch Cardiovasc Dis. 2010;103:454–459.
13. Shammas MA. Telomeres, lifestyle, cancer, and aging. Curr Opin Clin Nutr Metab Care. 2011;14:28–34.
14. Kyo S, Takakura M, Tanaka M, et al.. Expression of telomerase activity in human chorion. Biochem Biophys Res Commun. 1997;241:498–503.
15. Hohaus S, Voso MT, Orta-La Barbera E, et al.. Telomerase activity in human hematopoietic progenitor cells. Haematologica. 1997;82:262–268.
16. Akkad A, Hastings R, Konje JC, et al.. Telomere length in small-forgestational-age babies. BJOG. 2006;113:318–323.
17. Nordfjall K, Svenson U, Norrback KF, et al.. Large-scale parent-child comparison confirms a strong paternal influence on telomere length. Eur J Hum Genet. 2010;18:385–389.
18. Forraz N, McGuckin CP. The umbilical cord: a rich and ethical stem cell source to advance regenerative medicine. Cell Prolif. 2011;44(suppl 1):60–69.
19. Mold JE, Venkatasubrahmanyam S, Burt TD, et al.. Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans. Science. 2010;330:1695–1699.
20. Mehra R, Moore BA, Crothers K, et al.. The association between marijuana smoking and lung cancer: a systematic review. Arch Intern Med. 2006;166:1359–1367.
21. Kovacic P. Role of oxidative metabolites of cocaine in toxicity and addiction: oxidative stress and electron transfer. Med Hypotheses. 2005;64:350–356.
22. Yamamoto BK, Moszczynska A, Gudelsky GA. Amphetamine toxicities: classical and emerging mechanisms. Ann N Y Acad Sci. 2010;1187:101–121.
23. Oikawa S, Kawanishi S. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening. FEBS Lett. 1999;453:365–368.
24. Akiyama M, Hideshima T, Hayashi T, et al.. Nuclear factor-kappaB p65 mediates tumor necrosis factor alpha-induced nuclear translocation of telomerase reverse transcriptase protein. Cancer Res. 2003;63:18–21.
25. Takakura M, Kyo S, Inoue M, et al.. Function of AP-1 in transcription of the telomerase reverse transcriptase gene (TERT) in human and mouse cells. Mol Cell Biol. 2005;25:8037–8043.
26. Cong Y, Shay JW. Actions of human telomerase beyond telomeres. Cell Res. 2008;18:725–732.
27. Huang J, Okuka M, McLean M, et al.. Effects of cigarette smoke on fertilization and embryo development in vivo. Fertil Steril. 2009;92:1456–1465.
28. Izutsu T, Kudo T, Sato T, et al.. Telomerase activity in human chorionic villi and placenta determined by TRAP and in situ TRAP assay. Placenta. 1998;19:613–618.
29. Davy P, Nagata M, Bullard P, et al.. Fetal growth restriction is associated with accelerated telomere shortening and increased expression of cell senescence markers in the placenta. Placenta. 2009;30:539–542.
30. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature. 1990;345:458–460.
31. Hastie ND, Dempster M, Dunlop MG, et al.. Telomere reduction in human colorectal carcinoma and with ageing. Nature. 1990;346:866–868.
32. Allsopp R, Shimoda J, Easa D, et al.. Long telomeres in the mature human placenta. Placenta. 2007;28:324–327.
33. Bestilny LJ, Gill MJ, Mody CH, et al.. Accelerated replicative senescence of the peripheral immune system induced by HIV infection. AIDS. 2000;14:771–780.
34. Das B, Saini D, Seshadri M. Telomere length in human adults and high level natural background radiation. PLoS One. 2009;4:e8440.
35. De Meyer T, Rietzschel ER, De Buyzere ML, et al.. Paternal age at birth is an important determinant of offspring telomere length. Hum Mol Genet. 2007;16:3097–3102.
36. Kimura M, Cherkas LF, Kato BS, et al.. Offspring's leukocyte telomere length, paternal age, and telomere elongation in sperm. PLoS Genet. 2008;4:e37.
37. Friedrich U, Schwab M, Griese EU, et al.. Telomeres in neonates: new insights in fetal hematopoiesis. Pediatr Res. 2001;49:252–256.
38. Cross JA, Temple RC, Hughes JC, et al.. Cord blood telomere length, telomerase activity and inflammatory markers in pregnancies in women with diabetes or gestational diabetes. Diabet Med. 2010;27:1264–1270.
39. Blanche S, Tardieu M, Benhammou V, et al.. Mitochondrial dysfunction following perinatal exposure to nucleoside analogues. AIDS. 2006;20:1685–1690.
40. Blanche S, Tardieu M, Rustin P, et al.. Persistent mitochondrial dysfunction and perinatal exposure to antiretroviral nucleoside analogues. Lancet. 1999;354:1084–1089.
41. Brogly SB, Ylitalo N, Mofenson LM, et al.. In utero nucleoside reverse transcriptase inhibitor exposure and signs of possible mitochondrial dysfunction in HIV-uninfected children. AIDS. 2007;21:929–938.
42. Tardieu M, Brunelle F, Raybaud C, et al.. Cerebral MR imaging in uninfected children born to HIV-seropositive mothers and perinatally exposed to zidovudine. AJNR Am J Neuroradiol. 2005;26:695–701.
leukocyte telomere length; antiretroviral therapy; blood; HIV-infected pregnant women; HIV-exposed uninfected infants; HIV pregnancy
Supplemental Digital Content
© 2012 Lippincott Williams & Wilkins, Inc.