The effect of treatment was modeled, distinguishing between the effect of zidovudine alone and the effect of an association of antiretroviral drugs, and considering three periods: 0 to 6 weeks (during treatment), 6 weeks to 15 months and 15 months to 18 months to identify a possible late effect of perinatal drug exposure (Table 4). During the period from 0 to 6 weeks, infants exposed to zidovudine had significantly lower levels of neutrophils and lymphocytes than did untreated infants (neutrophils: −0.442, P < 0.0001; lymphocytes: −0.160, P < 0.0001). A similar, but non-significant trend was observed for platelets (−0.013, P = 0.28). During the other two periods (6 weeks to 15 months and 15 months to 18 months), levels of neutrophils, lymphocytes and platelets were consistently significantly lower in infants treated with zidovudine than in untreated infants.
For the periods from 0 to 6 weeks and from 6 weeks to 15 months, infants exposed to a combination of drugs had lower counts for all three cell lineages than did infants exposed to zidovudine monotherapy (Table 4). At 12 months, for example, the difference between untreated and monotherapy groups was 192 × 106 cells/l for neutrophils whereas that between untreated and combination therapy groups was 317 × 106 cells/l; the corresponding values for lymphocytes were 316 and 510 × 106 cells/l, and for platelets 10 × 109 and 24 × 109 (Table 5). To take into account other cofactors, these data were obtained from the modelization in the subgroup of non-premature boys born to non-African and non-drug-addicted mothers.
In general, the CD4+ and CD8+ lymphocyte subpopulations followed similar trends. The effects of treatment were greater on CD8+ than on CD4+ lymphocyte levels: the CD4+ lymphocyte levels were significantly decreased only until 15 months. These two variables showed no greater effect due to a combination of antiretroviral drugs than to monotherapy. Consequently, the findings for CD4+ and CD8+ lymphocytes are presented for all treated infants as a single group (Table 6). At 12 months, treated children had 144 × 106 fewer CD4 cells/l and 141 × 106 fewer CD8+ cells/l than untreated children.
A model including all the treated infants and the factors sex, geographical origin, prematurity, active maternal drug use and total duration of treatment revealed a significant negative relationship between treatment duration and neutrophil levels (−0.00025, P = 0.04), and treatment duration and lymphocyte levels (−0.00051, P < 0.0001). A similar, but non-significant trend was observed for platelets (−0.00008, P = 0.09).
We applied the same model, with the same factors, to the subgroup of infants for whom maternal CD4 cell counts at delivery were available (Table 7). The infants were assigned to three groups according to usual CD4 cell count thresholds (CD4 < 250 × 106 cells/l, 250 ≤ CD4 < 500 × 106 cells/l and CD4 ≥ 500 × 106 cells/l, the reference class). Total lymphocyte counts and particularly the CD4+ lymphocyte subset counts were significantly lower in the two groups of infants whose mothers had CD4 counts below 500 × 106 cells/l than in the group of infants whose mothers had CD4 counts of 500 × 106 cells/l or more. A similar trend was also noted for other lineages.
Observational studies of the effects of a drug in a cohort do not have the same rigor as a randomized study. Indeed, only a randomized study allows effects to be definitively attributed to the drug. However, the number of patients in randomized studies may often be too small to detect rare events, and effects on measures with large inter- and intra-individual variability. A large observational cohort may provide a sufficiently large number of subjects, but interpretation biases are possible. There are several factors that may be involved in effects on hematopoesis. Some are known, and can be included in multivariate analyses, but there may well be other as yet unidentified factors. Using mixed effects models to take into account the substantial variability of hematopoïetic variables, we first confirm what is already known, or widely suggested although rarely studied on so large a scale, concerning maternal geographic origin and sex: (1) total lymphocyte, CD4+, CD8+ and polynuclear neutrophil counts in infants with mothers of sub-Saharan African or Caribbean origin are lower than those in infants of European origin ; and (2) girls have higher levels of polynuclear neutrophils, lymphocytes and circulating platelets than boys [17–19].
More surprising is the relationship between the immune status of the mother at delivery and the lymphocyte count in the child. This relationship has not previously been described, to our knowledge, and may result from defective transplacental transfer of hematopoietic cytokines  in women with cellular immune deficiency. Consequences of maternal HIV infection in HIV uninfected babies have been recently evoked in a report of cardiac dysfunction in HIV-exposed but uninfected children .
The initial protocol comparing zidovudine and placebo revealed only an effect on hemoglobin levels that was reversed by the age of 12 weeks [1,4,5]. Our analysis reproduces this finding. The small but significant and persistent impact of perinatal prophylactic antiretroviral treatment on platelets, neutrophils and lymphocytes was not expected. The relatively small number of infants included in the previous analysis and the substantial inter- and intra-individual variability of hematopoietic variables may explain this discrepancy. The difference remained significant in a multivariate analysis, taking into account the factors listed above. Moreover, the greater effect of combination therapy than monotherapy and the link between the duration of exposure and the magnitude of the biological effect strongly implicate the treatment. The effect involves three hematopoiesis lineage and persists until 18 months of age despite the treatment having stopped at 6 weeks of age. An effect on several cell lineages suggests either changes in the medullary stroma or an effect on multipotent stem cells. There is no evidence suggesting that zidovudine has a deleterious effect on the medullary stroma. In contrast, the toxicity of zidovudine to hematopoietic progenitors in vitro has been well established [22,23]. The mechanism underlying this putative toxicity is unknown. The mitochondrial toxicity of antiretroviral nucleoside analogs is currently receiving considerable attention . However, non-mitochondrial mechanisms of toxicity are possible, mediated in particular by toxicity to nuclear DNA. Zidovudine interacts with nuclear DNA in several cell models , and the integration of zidovudine into nuclear DNA has been demonstrated both in an animal model  and in exposed newborns . More specifically, in vitro incorporation of zidovudine into DNA of burst-forming unit-erythroid (BFU.E) and colony-forming unit-erythroid (CFU-E) has been demonstrated  and leads to a reduced expression of the genes for β globin  or granulocyte-monocyte colony stimulating factor receptor (GM CSFR) . Although not addressed in this study, the clinical consequences of these findings are probably minor or non-existent at these ages. The observed deficiencies in lymphocyte, neutrophil and platelets lineages are clinically modest and no subgroup of patients with very low values was identified among the treated children. As suggested by the Danish study , a more detailed analysis of CD4/CD8 lymphocyte subpopulations (i.e naive/memory phenotype and function) could be of value, as would a more long-term evaluation. Indeed, our findings demonstrate that perinatal exposure to zidovudine may result in biological effects, even in a cell system with a high turnover, that persist until the age of 18 months.
This work was supported by Agence Nationale de Recherches sur le Sida (ANRS).
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The main investigator at each site and institutions participating in the French Perinatal Cohort Study
Coordinators: M.J. Mayaux and S. Blanche.
Aix-en-Provence, Thevenieau D.; Amiens, Pautard B.; Angers, Chennebault J.M.; Argenteuil, Allizy C.; Basse-Terre, Sibille G.; Bastia, Pincemaille O.; Bayonne, Hernandorena X.; Besançon, Estavoyer J.M.; Bondy, Lachassinne E.; Bordeaux, Douard D.; Boulogne Billancourt, Gilles I.; Bourg La Reine, Gantzer A.; Bullion, Colin-Gorki A.M.; Caen, Brouard J.; Cayenne, Delattre P.; Clamart, Vial M.; Clichy, Mazy F.; Colombes, Floch-Tudal C.; Compiègne, Lagrue A.; Corbeil Essonnes, Devidas A.; Creil, Duval-Arnould M.; Creteil, Touboul C.; Dijon, Guerin M.N.; Dourdan, Ercoli V.; Dreux, Denavit M.F.; Elbeuf, Lahsinat K.; Evreux, Pascal C.; Evry, May A.; Fontainebleau, Dallot M.C.; Fort de France, Cecile W.; Gonesse, Lobut J.B.; Lagny sur Marne, Chalvon Dermesay A.; Le Chesnay, Beal G.; Le Kremlin Bicêtre, Bader-Meunier B.; Le Lamentin, Monlouis M.; Lille, Mazingue F.; Limoges, De Lumley L.; Longjumeau, Seaume H.; Lyon, Kebaili K.; Mantes La Jolie, Botto C.; Marseille, Thuret I.; Meaux, Crumiere C.; Melun, Le Lorier B.; Meulan, Seguy D.; Montfermeil, Talon P.; Montpellier, Nicolas J.; Montreuil, Heller-Roussin B.; Nancy, Hubert C. L.,; Nanterre, De Sarcus B.; Nantes, Mechinaud F.; Neuilly sur Seine, Berterottiere D.; Nice, Monpoux F.; Nîmes, Dendale J.; Orléans, Arsac P.; Orsay, De Gennes C.; Perpignan, Bachelard G.; Pointe-à-Pitre, Bardinet F.; Poissy, Rousset M.C.; Pontoise, Mouchnino G.; Reims, Munzer M.; Rouen, Brossard V.; Saint-Denis, Retbi J.M.; Saint-Etienne, Fresard A.; Saint-Germain en Laye, Narcy P.; Saint-Martin, De Caunes F.; Sèvres, Segard L.; Strasbourg, Partisani M.; Suresnes, Clement; Toulouse, Tricoire J.; Tours, Marchand S.; La Trinité, Hugon N.; Villeneuve Saint Georges, Guillot F.; Villepinte, Broyard A.
Paris: ASE St Vincent de Paul, Commeau A.; Hospitalier Cochin Tarnier Port-Royal, Firtion G.; Groupe Hospitalier Necker, Blanche S., Parat S.; Hôpital Bichat-Claude Bernard, Matheron S.; Hôpital des Métallurgistes, Heller-Roussin B.; Hôpital La Pitié Salpétrière, Noseda G.; Hôpital Lariboisière, Ciraru-Vigneron N.; Hôpital Notre Dame de Bon Secours, Ayral D.; Hôpital Robert Debré, Levine M.; Hôpital Rothschild, Wallet A.; Hôpital Saint-Antoine, Carbonne B.; Hôpital Saint-Michel, Aufrant C.; Hôpital Saint-Vincent de Paul, Boccara J.F.; Hôpital Tenon, Herve F.; Hôpital Trousseau, Dollfus C.; Institut de Puériculture Brune, Dubois M.; Institut Mutualiste Montsouris, Carlus Moncomble C.