HIV protease inhibitor use during pregnancy is associated with decreased progesterone levels, suggesting a potential mechanism contributing to fetal growth restriction

Abstract

Background: Protease inhibitor (PI)-based combination antiretroviral therapy (cART) is administered during pregnancy to prevent perinatal human immunodeficiency virus (HIV) transmission. However, PI use has been associated with adverse birth outcomes, including preterm delivery and small-for-gestational-age (SGA) births. The mechanisms underlying these outcomes are unknown. We hypothesized that PIs contribute to these adverse events by altering progesterone levels.

Methods: PI effects on trophoblast progesterone production were assessed in vitro. A mouse pregnancy model was used to assess the impact of PI-based cART on pregnancy outcomes and progesterone levels in vivo. Progesterone levels were assessed in plasma specimens from 27 HIV-infected and 17 HIV-uninfected pregnant women.

Results: PIs (ritonavir, lopinavir, and atazanavir) but not nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors reduced trophoblast progesterone production in vitro. In pregnant mice, PI-based cART but not dual-NRTI therapy was associated with significantly lower progesterone levels that directly correlated with fetal weight. Progesterone supplementation resulted in a significant improvement in fetal weight. We observed lower progesterone levels and smaller infants in HIV-infected women receiving PI-based cART, compared with the control group. In HIV-infected women, progesterone levels correlated significantly with birth weight percentile.

Conclusions: Our data suggest that PI use in pregnancy may lead to lower progesterone levels that could contribute to adverse birth outcomes.

Keywords: HIV; lopinavir; low birth weight; pregnancy; progesterone; protease inhibitors; small for gestational age.

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

Figures

Figure 1.

Protease inhibitors decrease trophoblast progesterone production in vitro. A, BeWo cells, a human cytotrophoblast cell line, were treated for 24 hours with 10 times the minimal effective dose (see Methods) of zidovudine (ZDV), lamivudine (3TC), nevirapine (NVP), ritonavir (RTV), atazanavir (ATV), darunavir (DRV), or lopinavir (LPV). B, BeWo cells were treated for 24 hours with combinations of ZDV plus 3TC and RTV-boosted ATV (ATV/r), DRV (DRV/r), or LPV (LPV/r). Control cells (Ctr) were treated with dimethyl sulfoxide at a final concentration of <0.1%. Cells incubated in hypoxic conditions (1% oxygen; Hyp) were used as a positive control. Progesterone levels were measured by competitive enzyme immunoassay and were corrected for the number of cells per well. Progesterone levels are expressed as percentage of the median control value. Data displayed are means±standard errors of the mean for 3–6 independent experiments. *P < .05 and **P < .01 for comparisons of each value with the control, by analysis of variance with the Dunnett post hoc test.

Figure 2.

Mouse plasma progesterone levels are decreased in protease inhibitor (PI)–exposed pregnant mice and correlated with fetal weight. Mated mice were exposed to either Combivir alone (zidovudine plus lamivudine; dual nucleoside reverse transcriptase inhibitor [NRTI]), Combivir plus Kaletra (ritonavir-boosted lopinavir; PI based combination antiretroviral therapy [PI-cART]), or water as a control (Ctr) by gavage once daily starting on gestational day 1. A, The percentage of fetuses that were viable (light grey), nonviable (as assessed by pedal reflex) (white), or resorbed (dark grey) for each treatment group is shown. χ2 analysis yielded the following findings: Ctr vs PI-cART, P < .001; dual NRTI vs PI-cART, P < .01; and Ctr vs dual NRTI, P = not significant. B, Average fetal weight per litter. C, Average placenta weight per litter. D, Progesterone levels in maternal plasma. Levels normalized to the control median are shown. In panels B, C, and D, data are shown as box and whisker plots, with medians, interquartile ranges, and ranges. Statistical comparisons were assessed by the Kruskal–Wallis test with the Dunn post hoc test. Data in panels A–D were acquired from the same experiment with 10 values for the Ctr group, 8 for the PI-cART group, and 8 for the dual NRTI group. Experiments were repeated 2 times. *P < .05, **P < .01, and ***P < .001. E, Progesterone levels were plotted against the average fetal weight per litter for the PI-cART group. Correlation was assessed by Spearman r. Trend line was calculated by linear regression analysis. Data represent 17 values from 3 combined experiments.

Figure 3.

Progesterone supplementation partially reverses protease inhibitor (PI)–induced fetal weight defect. Mated mice were exposed to Combivir plus Kaletra (PI based combination antiretroviral therapy [PI-cART]) or water as a control (Ctr) by gavage once daily starting on gestational day 1 (GD1). Mice were then injected subcutaneously with either 0.5 mg progesterone (P4) suspended in corn oil or corn oil as a control on GD1, GD5, GD9, and GD13. All fetal and placenta parameters were assessed on GD15. A, Percentage of fetuses that were viable (light grey), nonviable (as assessed by pedal reflex; white), or resorbed (dark grey) for each treatment group is shown. χ2 analysis yielded the following findings: Ctr vs PI-cART, P < .001; PI-cART vs PI-cART + P4, P = not significant; and Ctr vs PI-cART + P4, P < .05. B, Average fetal weight per litter. C, Average placental weight per litter. Data were acquired from the same experiment, with 8 values for the Ctr, 10 for the PI-cART group, and 11 for the PI-cART + P4 group. Experiments were repeated once. *P < .05, **P < .01, and ***P < .001, by the Kruskal–Wallis test with the Dunn post hoc test.

Figure 4.

Progesterone levels are lower in protease inhibitor (PI)–exposed human immunodeficiency virus (HIV)–infected pregnant women and correlated with birth weight percentile. A, Plasma progesterone assessed by enzyme immunoassay in plasma specimens obtained during gestational weeks (GW) 25–28 from 17 HIV-negative and 27 HIV-infected pregnant women. B, Progesterone levels in 22 HIV-infected women receiving PI-based combination antiretroviral therapy (cART) and 5 receiving nonnucleoside reverse transcriptase inhibitor (NNRTI)–based cART. C, Correlation between birth weight percentile and progesterone levels in 22 HIV-infected women receiving PI-based cART. All data are shown as scatterplots with median values. Statistical comparisons were made using the Mann–Whitney test. Correlation was assessed by Spearman r.