No association between early antiretroviral therapy during pregnancy and plasma levels of angiogenic factors: a cohort study

Background Early antiretroviral therapy (ART) during pregnancy has dramatically reduced the risk of perinatal HIV transmission. However, studies have shown an association between premature delivery and the use of ART during pregnancy (particularly protease inhibitor (PI)-based therapies), which could be explained by placental dysfunction. The objective of this study was to evaluate the association of ART (class, duration of exposure and time of initiation) with placental function by using angiogenic factors placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) as biomarkers. Methods Clinical and biological data from 159 pregnant women living with HIV were analyzed. Levels of each biomarker were measured in the first and second trimester of pregnancy. After logarithmic transformation, we compared these using generalized estimating equations according to (a) the type of ART; (b) the duration of exposure to ART; and (c) the time of initiation of ART. Results After adjusting for variables such as ethnicity, maternal age, gestational age, body mass index, parity, smoking status, and sex of the fetus, we found no significant association between the class of ART (PI-based or not) and serum concentrations of PlGF or sFlt-1. Furthermore, no significant association was found between biomarker levels and the duration of ART exposure or the timing of ART initiation (pre- or post-conception). Conclusions This study suggests that first and second trimester angiogenic factor levels are not significantly associated with ART, regardless of the duration or type (with or without PI). These observations seem reassuring when considering the use of ART during early pregnancy.

these women want to have children.(2) Antiretroviral therapy (ART) reduces the risk of HIV transmission from mother to child to 1-2%, compared to 15-40% without intervention. (3) ART also improves the health status of the mothers, raises their quality of life and prolongs their life expectancy, leading more and more women living with HIV to conceive.
(2) According to current recommendations, all adults living with HIV should be treated with ART.(4) As a result, most HIV-infected women are now already on ART at the time of conception and early pregnancy, whereas in the past, ART was initiated only in the second trimester of pregnancy. (5) Although ART has significantly reduced rates of adverse perinatal outcomes such as stillbirth, intrauterine growth restriction and preterm birth associated with maternal HIV infection,(6, 7) these rates are still higher for women living with HIV while receiving ART compared to HIV-negative women.(8-11) These adverse pregnancy outcomes may be related to the class of ART used during pregnancy, notably to protease inhibitor (PI) regimens, (11)(12)(13)(14)(15) which have been associated with placental vascular changes (16).
Anti-angiogenic effects have been reported following the use of PIs in oncology. (17,18) Placental growth factor (PlGF) and the soluble receptor, soluble fms-like tyrosine kinase-1 (sFlt-1) are respectively pro-and anti-angiogenic factors. PlGF is synthesized in several organs (heart, skeletal muscle, lungs, adipose tissue, platelets). sFlt-1 is released by vascular endothelial cells and circulating cells (monocytes, macrophages, platelets). The placenta (trophoblast and endothelial cells of the placental villi) is the main source of PlGF and sFlt-1 during pregnancy; they participate in vasculogenesis and feto-placental angiogenesis. Increased sFlt-1 and decreased free PlGF levels in maternal blood, or increased sFlt-1/ PlGF ratio are directly implicated in the pathophysiology of preeclampsia, including maternal endothelial dysfunction. (19)(20)(21)(22)(23)(24)(25)(26) A change in these factors is also associated with other complications during pregnancy, such as intrauterine growth restriction,(24, 27) preterm delivery,(28) spontaneous abortion and stillbirth, (23,29,30) This study included data from pregnant women who were enrolled between January 2003 and December 2016, and for whom serum samples were available during both the first trimester (5-14 weeks) and the second trimester (15-28 weeks) of pregnancy. Maternal serum samples were collected at the time of clinically indicated blood tests and stored at -80°C for research purpose. Levels of PlGF and sFlt-1 were measured using DuoSet Enzyme-Linked Immunosorbent Assay (ELISA) kits (R&D systems, Minneapolis, MN). Lower limits of detection were 15.6 pg/ml (sFlt-1) and 62.5 pg/ml (PlGF). There was no dilution for PlGF and it is a factor of 5 for sFLT-1. In all cases, both biomarkers were assayed using the same serum aliquot.
Gestational age was defined based on the crown-rump length from the first-trimester ultrasound if available and if not, from the date of the last menstrual period. Preterm birth was defined as delivery before 37 weeks of gestation. For gestational age, small (SGA) was defined as a birth weight below the 10th percentile for the gestational age.
Women living with HIV were categorized according to ART exposure at first and second trimester (PI-based ART, other ART, or no treatment). The duration of ART exposure during pregnancy was expressed in weeks since conception. Time of initiation of ART was defined relative to conception (either before conception or during pregnancy).

Statistical analysis
Descriptive analyses were conducted on the socio-demographic, clinical and biological data of the participants. For each categorical and continuous variable, data are reported as proportions or mean (with standard deviation) or median with interquartile range (IQR) respectively. The Wilcoxon test for matched samples was used to compare serum marker concentrations in the first and second trimesters and Mann-Withney U test to compare angiogenic factor levels in the two groups with undetectable viral load or not.
Linear regression evaluated the association between angiogenic factor concentrations and birth outcome groups (preterm birth and SGA) at the first and second trimester. To account for repeated measurements from the same individuals in the first and second trimesters of pregnancy, linear generalized estimating equations (GEE) were used to analyze the association between ART (class, duration of exposure and initiation time) and plasma concentration of the two biomarkers. A first-order autoregressive (AR1) correlation matrix was used. Models were adjusted for potential confounding factors previously  Table 1. In the first trimester, nearly 69% of women were receiving ART, 82% of whom were receiving PI-based regimens. In the second trimester, more than 96% were receiving ART, 87% of which were PI-based. The mean duration of ART exposure from the start of pregnancy to first-trimester and second-trimester biomarker testing was 9.5 ± 2.9 weeks and 14.8 ± 7.1 weeks, respectively.     Table 2. After adjustment, no significant association was found between the class of ART (whether PI-based or not) and the level of PlGF, sFlt-1 or the PlGF/SFlt-1 ratio ( Table 2). Furthermore, no significant association was found between serum angiogenic factors and duration of ART exposure (including all PIcontaining ART) or initiation time (pre-conception versus during pregnancy) ( Table 2).

Table 2. Association between biomarkers and ART (type, duration and initiation time
The association between angiogenic factor concentrations and birth outcomes (preterm birth and SGA) was further evaluated (Table 3). After adjustment, significantly lower concentrations of sFlt-1 and sFlt/PlGF ratio at the first trimester and significantly lower concentrations of PlGF at the second trimester were seen in SGA cases than in normal weight cases. No significant association between angiogenic factor concentration and preterm birth was observed.

Discussion
In this cohort study, first and second trimester angiogenic factor concentrations are not significantly associated with ART exposure of any type and duration, nor are they associated with the timing of treatment initiation (pre-conception or during pregnancy).
The biomarker levels observed among women living with HIV appear to be similar to those reported in HIV-infected women in other studies.(41-45) The strong association of these angiogenic factor concentrations with gestational age and other baseline data (BMI, ethnicity) is consistent with data from HIV-negative pregnant women.(45, 46) This explains why the significant differences between ART exposure groups in the unadjusted analyses were not confirmed in the multivariate analyses ( Table 2).
The absence of an HIV-negative control group in this study is a limitation that prevents us from assessing the impact of HIV infection itself on angiogenic factors and whether the infection itself can lead to dysregulation of angiogenesis. However, it seems unlikely that viral activity has much of an influence on angiogenic factor concentrations when we consider the similarity of levels in the untreated group (with a detectable viral load) compared to the treated one. The heterogeneity of ART regimens received by women is another limitation of our study (see Table, Additional file 1, which illustrates the different nucleoside reverse transcriptase inhibitors in the ART). However, repeated measurements in the first and second trimester increased the number of observations, giving us a greater statistical power. Measurements of the levels of angiogenic factors were all performed on the same day to minimize inter-assay variability. We also attempted to eliminate bias with a conservative method of adjustment for potential confounding factors.
Very few studies have explored the relationship between ART and the serum concentration of these angiogenic factors during pregnancy. Studies in oncology, however, have suggested anti-angiogenic effects for some PIs. (17,18) The effects of PIs on placental vascular system formation and fetal development have to date only been examined in a mouse model.(47) Mice exposed to ART had significantly smaller fetuses and placentas compared to controls. Litter size and fetal viability were negatively impacted by exposure to two nucleoside analogs and two PIs at doses equivalent to human therapeutic doses.
Although PlGF levels were unchanged, significantly lower levels of placental sFlt-1 were noted. (16,47) Lower PlGF levels were reported in South Africa amongst pregnant women living with HIV, compared to uninfected pregnant women, whether or not they were preeclamptic. To our knowledge, our study constitutes the first report on first-trimester angiogenic marker levels in a cohort of women who received early exposure to ART during pregnancy.
Our results are consistent with reports on later ART initiation in pregnancy.(34) These finding needs to be confirmed by the study of circulating levels of other angiogenic biomarkers as well as direct study of the early placenta vasculature. If confirmed, even though angiogenic processes in the placenta are critical regulators of fetal growth and impact birth outcomes, the pathophysiological mechanism of the association between ART and preterm birth would unlikely be through an early and direct effect on placental angiogenesis. Immune restoration as a result of ART initiation is a hypothesis that needs to be explored.

Conclusions
This study suggests that ART, whether PI-based or not, is not associated with the serum concentration of angiogenic factors PlGF and sFlt-1 in the first and second trimesters of pregnancy. There is also no significant association with duration of treatment or timing of treatment initiation (before conception or during pregnancy). These observations seem generally reassuring for the potential consequences of early ART use during pregnancy.
Further studies are needed to confirm the safety of early ART exposure regarding placental angiogenesis and its implications for adverse pregnancy outcomes.

Availability of data and material
The datasets generated and/or analysed during the current study are not publicly available due to restrictions associated with anonymity of participants but are available from the corresponding author on reasonable request.       additional file 1.pdf