Lipid based nutrient supplements during pregnancy may improve foetal growth in HIV infected women : A cohort study

RESEARCH ARTICLE Lipid based nutrient supplements during pregnancy may improve foetal growth in HIV infected women – A cohort study Minyanga Nkhoma 1 , Per Ashorn 1,2 , Ulla Ashorn 1 , Kathryn G. Dewey 3 , Austrida Gondwe 1 , Kenneth Maleta 4 * 1Centre for Child Health Research, Faculty of Medicine and Health Technology, Tampere, University, Tampere, Finland, 2Department of Paediatrics, Tampere University Hospital, Tampere, Finland, 3Department of Nutrition, University of California Davis, Davis, California, United States of America, 4School of Public Health and Family Medicine, University of Malawi College of Medicine, Blantyre, Malawi *[email protected]w Abstract Objectives Both maternal HIV infection and antiretroviral therapy are associated with adverse birth outcomes. The role of antenatal nutrient supplements with regard to adverse birth outcomes in HIV infected women exposed to antiretroviral therapy is not well known. We assessed the association between HIV and birth outcomes and explored whether antenatal lipid-based nutrient supplements (LNS) modulated this association. Methods We analysed a nested cohort of pregnant Malawian women who received daily LNS, multiple micronutrients (MMN) or iron and folic acid (IFA). Birth weight, length-for-age z-score (LAZ) and weight-for-age z-score (WAZ) were analysed as continuous outcomes and proportion of stunting and small-for-gestational age (SGA) as dichotomous outcomes. Results 134 HIV infected (46 LNS, 39 MMN, 49 IFA) and 833 HIV uninfected (271 LNS, 287 MMN, 275 IFA) women were included. Maternal HIV infection was associated with a lower mean birth weight (-129g (-209, -48), P = 0.002); LAZ (-0.34 (-0.54, -0.13), P = 0.002) and WAZ (-0.21 (-0.40, -0.02), P = 0.041) and a higher risk of stunting (RR (95% confidence interval), 1.87 (1.24, 2.83), P = 0.003) and SGA (1.66 (1.21, 2.26), P = 0.001) in the newborn. If the women received LNS, HIV was not associated with LAZ (mean difference (95%); -0.02 (-0.35, 0.31), P = 0.918) or newborn stunting (RR (95% CI), 0.84 (0.34, 2.03), P = 0.691). However HIV tended to be associated with LAZ if the women received MMN (-0.42 (-0.80, -0.03), P = 0.053); and was significantly associated with LAZ if the women received IFA (-0.52 (-0.89, -0.14), P = 0.021) and with newborn stunting if they received MMN (2.40 (1.15, 4.98), P = 0.029) or IFA (2.40 (1.26, 4.59), P = 0.024). PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 1 / 15 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Nkhoma M, Ashorn P, Ashorn U, Dewey KG, Gondwe A, Maleta K (2019) Lipid based nutrient supplements during pregnancy may improve foetal growth in HIV infected women – A cohort study. PLoS ONE 14(5): e0215760. https:// doi.org/10.1371/journal.pone.0215760 Editor: Karel Allegaert, KU Leuven, BELGIUM Received: December 7, 2018 Accepted: April 7, 2019 Published: May 2, 2019 Copyright: ©2019 Nkhoma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The de-identified, individual-level minimal underlying data set has been deposited to Open Science Framework: https://osf.io/UX7PT/. Funding: This publication is based on research funded in part by the Office of Health, Infectious Diseases, and Nutrition, Bureau for Global Health, U.S. Agency for International Development (USAID) under terms of Cooperative Agreement No. AID-OAA-A-12-00005, through the Food and Nutrition Technical Assistance III Project (FANTA), managed by FHI 360, with additional funding from Conclusions Further research to investigate the impact of LNS on various aspects of foetal growth in HIV infected women is warranted. Introduction Global estimates indicate that 17.8 million women aged >15 years were living with human immunodeficiency virus (HIV) in 2016 [1]. Following the rapid scale-up of antiretroviral therapy (ART) [2] there has been a considerable reduction in vertical transmission and HIV associated maternal and child mortality [1]. However, maternal HIV continues to be associated with adverse birth outcomes such as preterm birth (PTB) and low birth weight (LBW) even in the context of ART use [3]. Indeed, different aspects of maternal ART including timing of initiation of therapy (pre-conception vs post-conception) or type of antiretroviral regimen have been reported to influence the risk of adverse birth outcomes in HIV infected women [4,5]. Mechanisms for PTB or foetal growth restriction (FGR) in HIV infected women are not well understood. Maternal anaemia, low body-mass-index (BMI), low mid upper arm circumference (MUAC) and low gestational weight gain (GWG) have been identified as risk factors for PTB and FGR in HIV infected women [6–8]. A possible pathway for PTB and FGR is increased oxidative stress levels that have been observed more frequently in HIV infected individuals [9] and in placentas from HIV infected women [10]. Oxidative stress has been linked to PTB and FGR [11,12]. Considering this knowledge, antenatal provision of dietary supplements that may improve maternal nutritional status or reduce oxidative stress may prevent the occurrence of these adverse birth outcomes. In this study, we evaluated the association between maternal HIV infection and the rate of gestational weight gain (GWG), inadequate GWG and a wide range of birth outcomes (duration of pregnancy, birth weight, length-for age z-score (LAZ), weight-for-age z-score (WAZ), weight-for-length z-score (WLZ), head circumference-for-age z-score (HCZ), PTB, LBW, stunting, underweight, wasting and small-for-gestational age (SGA)). We further examined whether the association between HIV and these pregnancy outcomes would be influenced by lipid-based nutrient supplements (LNS). LNS contain several micronutrients, macrominerals and macronutrients such as protein and essential fatty acids [13]. Alpha linolenic acid, an n-3 fatty acid, has been shown to reduce oxidative stress in rats [14]. Materials and methods We conducted a secondary cohort analysis of data collected from pregnant women who participated in the iLiNS-DYAD-Malawi trial (Trial Registration www.clinicaltrials.gov, Identifier: NCT01239693). This was a randomized controlled nutrient intervention trial that evaluated the impact of LNS on pregnancy outcomes. The analysis we are reporting now was defined a priori in the iLiNS-DYAD-Malawi study protocol. Ethical approval for the trial was obtained from the College of Medicine Research and Ethics Committee, University of Malawi and the Ethics Committee of Pirkanmaa Hospital District, Finland. Written informed consent was sought from all study participants prior to study participation. Details of the main iLiNS-DYAD-M trial and the main study results have been described elsewhere [15]. Briefly, we recruited pregnant women who attended antenatal clinics at four Lipid based nutrient supplements, maternal HIV infection and adverse pregnancy outcomes PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 2 / 15 a grant to the University of California, Davis by the Bill & Melinda Gates Foundation, OPP49817 to KGD. For data management and statistical analysis, the team received additional support in grants from the Academy of Finland (grant 252075) and the Medical Research Fund of Tampere University Hospital (grant 9 M004). The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the United States Government, USAID, the Bill & Melinda Gates Foundation, or the other funders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. health facilities in Mangochi district, a predominantly rural district in South Eastern Malawi. Provision of LNS was not associated with improved birth outcomes [15]. As part of the main trial, participating women received daily oral iron and folic acid (IFA) tablet, multiple micronutrients (MMN) tablet or a sachet of small-quantity LNS throughout pregnancy. The description of the study supplements [15] and the rationale for the formulation of LNS have been reported previously [13]. Briefly, the IFA tablets contained 60 mg of iron (standard dose) whereas the MMN tablets and the LNS sachet contained 20 mg iron each; all 3 supplements contained 400 mcg of folic acid. In addition to IFA the MMN and LNS supplements contained 16 other micronutrients. The LNS also contained 4 macrominerals (phosphorus, calcium, potassium and magnesium) and essential fatty acids (linoleic acid and alphalinolenic acid) and provided 118 kcal per day. For this report, we used data that were collected at enrolment (baseline and exposure data) and at two scheduled clinic visits at 32 and 36 gestation weeks (maternal weight), and as soon as possible after birth and at 1–6 weeks postpartum (newborn anthropometry). The outcome variables of interest were rate of GWG (g/ week), pregnancy duration (gestation weeks), birth weight (grams), LAZ, WAZ, WLZ, HCZ, inadequate GWG (GWG below the lower limit of the recommended weight gain according to the Institute of Medicine’s (IOM) recommendations [16]), PTB (delivery occurring <37 gestation weeks), LBW (birth weight <2500g), stunting (LAZ <-2 at birth) underweight (WAZ<-2 at birth), wasting (WLZ<-2), small head circumference (HCZ <-2 at birth) and SGA (birth weight below the 10th centile of the Intergrowth Standards). Maternal HIV infection at enrolment was the main exposure of interest. Based on prior axiomatic knowledge, the following baseline variables were defined a priori as potential confounders for the relationship between HIV and adverse pregnancy outcomes: maternal age (years), maternal education (number completed years in school), proxy score for socio-economic status (SES) estimated based on household assets adjusted score system [17], BMI (maternal weight (kg) / maternal height (cm) 2 ), MUAC (measured in cm to one decimal place), maternal anaemia at enrolment (haemoglobin <110g/l) and maternal Plasmodium falciparum infection at enrolment. The type of nutrient supplement received in the iLiNS-- DYAD-M trial was considered a potential effect modifier of the relationship between HIV infection and study outcomes. We determined maternal HIV status on venous blood using the Alere Determine HIV-1/2 Rapid Test (Alere Medical Co) and confirmed positive results using Uni-Gold Rapid Test (Trinity Biotech Co., Wicklow). Use of ART was variable among the HIV infected women who participated in the study. A small proportion of the HIV infected women who already knew their status were using ART prior to conception. Newly diagnosed women or those who were not yet on ART were referred to the health facility’s ART clinic for further management of HIV infection. We analysed our data using Stata 12.1 (StataCorp, College Station, USA). We restricted our analyses to women who had live singleton births only. Specifically, we excluded women who had no HIV test result, twin gestation, miscarriage, stillbirth, unusual data (gestational age >42 weeks), or incomplete outcome data. The rate of GWG was calculated as the difference between maternal weight measured at the 32/36-wk visit minus maternal weight measured at baseline divided by the number of weeks between these 2 measurements. We defined inadequate GWG as weekly GWG below the lower limit of the Institute of Medicine (IOM) recommended rate for the second and third trimesters (454, 364, 227, 182 g/week for underweight, normal-weight, overweight and obese women respectively) [16]. Since the IOM recommendations are based on pre-pregnancy BMI, we estimated maternal BMI before pregnancy by first determining the best transformation of maternal BMI at Lipid based nutrient supplements, maternal HIV infection and adverse pregnancy outcomes PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 3 / 15 enrolment that achieved a normal distribution and then regressed the transformed BMI with age, age squared, and age cubed to generate predicted and residual values. We then inspected the regression curve to determine the earliest gestation age before the confidence interval expanded, with the assumption that weight gain before that point was minimal. Next, we calculated the predicted mean BMI at 13.7 weeks, and this was used as a proxy for pre-pregnancy BMI. We compared continuous measurements and categorical outcomes between HIV infected and HIV uninfected participants, and between women included or excluded from the analysis. We used linear regression to estimate the coefficient or difference in means. We estimated the risk ratio for the comparison of dichotomous outcomes at a single time point using log binomial regression models or Poisson regression models in case the algorithm failed to converge in the estimation. To control for possible confounding, we adjusted for covariates that were identified a priori as being potentially associated with both HIV infection and FGR. We assessed heterogeneity of effects by stratifying HIV versus No-HIV comparisons by intervention group. We corrected for multiple testing using the false discovery method [18]. Because maternal HIV status was not listed as one of the main potential effect modifiers (hypothesis-driven) in the iLiNS-- DYAD-M statistical analysis plan, the stratified analyses were exploratory. Results Out of the 1391 women enrolled into the iLiNS-DYAD-M study, 967 were eligible for analysis. We performed our outcome analyses on this cohort of 134 HIV infected and 833 HIV uninfected pregnant women. Women excluded from the outcome analyses (n = 424) due to missing HIV test results, twin gestation, miscarriage, stillbirth, dropout or incomplete outcome data (Fig 1) tended to be younger (23.9 versus 25.4 y), to be wealthier (0.15 versus -0.05), to be primigravid (31.8% versus 17.5%), to have a higher BMI (22.5 versus 22.0 kg/m 2 ) and to be anaemic (24.4% versus 19.0%) but were otherwise similar to women included in the analyses in terms of the other baseline characteristics (S1 Table). Overall, the rate of attrition was similar between the HIV negative and HIV positive women (27.6 vs 26.8%, P = 0.801). The rates of miscarriage or stillbirth did not differ according to maternal HIV infection (P = 0.259; P = 0.341, respectively). Baseline characteristics of HIV Fig 1. Flow chart of participants included in the analysis. https://doi.org/10.1371/journal.pone.0215760.g001 Lipid based nutrient supplements, maternal HIV infection and adverse pregnancy outcomes PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 4 / 15 infected, and uninfected women were similar in terms of their height, BMI, education attainment and gestational age at enrolment (P>0.05). However, HIV infected participants tended to be older, to be wealthier, to have a lower haemoglobin level and were less likely to be primigravid (Table 1). HIV infected women did not differ significantly from HIV uninfected women in terms of pregnancy duration or newborn WLZ or HCZ, but they had a slower rate of GWG (273 vs 297 g/week, P = 0.004) and they delivered babies who were lighter (2882 versus 2987g, P = 0.002) and shorter (LAZ -1.28 versus -0.99, P = 0.002 (Table 2). The proportions of infants with PTB, LBW, stunting and SGA were significantly higher among HIV infected women as compared to HIV uninfected women. However, there were no statistically significant associations between maternal HIV status and proportion of women with inadequate GWG or proportion of infants with wasting, underweight or small head size (Table 3). The numbers randomised to receive LNS, MMN and IFA were 46, 39 and 49, and 271, 287 and 275 in the HIV infected and HIV uninfected women respectively. Stratified analyses for continuous birth outcomes that were significantly associated with HIV infection in the main analysis showed that the values for birth weight, LAZ and WAZ for HIV infected women who received LNS were consistently closer to those of uninfected women regardless of the type of the intervention received by the uninfected women. Comparisons between HIV infected and uninfected women indicated that for all outcomes the values were mostly lower among infected versus non-infected women in each of the intervention groups, though these differences were statistically significant only for birth weight in the MMN group and LAZ in the IFA group (Table 4). Like the findings from the stratified analyses of continuous outcomes, the risk of preterm birth, and stunting in HIV infected women who received LNS was closer to the risk observed in HIV uninfected women regardless of type of supplement received by the uninfected women. Maternal HIV was not associated with LBW or SGA in any of the three intervention groups. In contrast, the risk of PTB was significantly higher among infected versus uninfected women only among those in the MMN group and not in the IFA or LNS groups. The risk of Table 1. Baseline characteristics of the women included in the analysis, by HIV status. Characteristic No HIV HIV P value Number of participants 833 134 Mean (SD) maternal age, years 24.9 (6.1) 28.8 (5.2) <0.001 Mean (SD) maternal education, completed years at school 4.0 (3.4) 3.9 (3.6) 0.755 Mean (SD) household assets score -0.09 (0.92) 0.13 (1.10) 0.021 Mean (SD) gestational age at enrolment, weeks 16.8 (2.1) 16.6 (2.2) 0.075 Primigravid, % 19.8 3.0 <0.001 Mean (SD) height, cm 156.1 (5.6) 156.2 (5.9) 0.849 Mean (SD) MUAC, cm 26.3 (2.6) 26.6 (2.3) 0.105 Wasting (MUAC<22 cm), % 7.2 4.5 0.246 Mean (SD) BMI, kg/m 2 22.0 (2.8) 22.3 (2.5) 0.243 Low BMI (<18.5 kg/m 2 ), % 5.7 4.5 0.354 Mean (SD) blood hemoglobin concentration, g/l 112 (16.1) 108 (17.6) 0.002 Anaemia (Hb <100 g/l), % 18.3 23.9 0.125 P.falciparum infection (RDT), % 22.3 21.6 0.873 MUAC, mid-upper arm circumference; BMI, body-mass-index; RDT, rapid diagnostic testing P-values were obtained from t-test (comparison of means) or Chi square test (comparison of proportions). https://doi.org/10.1371/journal.pone.0215760.t001 Lipid based nutrient supplements, maternal HIV infection and adverse pregnancy outcomes PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 5 / 15 newborn stunting was significantly higher among infected versus uninfected women in the IFA and MMN groups but similar between infected and uninfected women who received LNS (Table 5). Out of the 134 HIV infected participants, 38 (28.4%) received ART during pregnancy (20 started ART prior to joining the study and 18 after enrolment into the study) and 10 (7.5%) started ART after delivery; for 30 (22.3%) women it was unknown when they started therapy and for 43 (32.1%) treatment status was unknown. ART status was not associated with type of nutrient supplement received in the main trial (P = 0.702). Because ART use during pregnancy may increase the risk of adverse birth outcomes [19], we conducted a sensitivity analysis in which we excluded all HIV positive women who were on ART (Tables 6and 7). The associations between maternal HIV and GWG, birth weight, LAZ, WAZ, preterm birth, LBW, newborn stunting and SGA persisted (P<0.05) after excluding those who were on ART. Notably, the associations between HIV and LBW, newborn stunting and SGA were Table 2. Continuous pregnancy outcomes by HIV infection status a . Outcome No HIV (N = 833) HIV (N = 134) Unadjusted difference in means (95% CI) b P-value b Adjusted difference in means (95% CI) b P-value b Gestational weight gain rate, g/ week 297 (102) 273 (108) -25 (-43, -6) 0.010 -28 (-47, -9) 0.004 Duration of pregnancy, weeks 39.4 (1.60) 39.3 (1.84) -0.11 (-0.41, 0.19) 0.470 -0.11 (-0.42, 0.20) 0.493 Birth weight, g 2987 (425) 2882 (447) -105 (-183, -27) 0.009 -129 (-209, -48) 0.002 Length-for-age z-score -0.99 (1.10) -1.27 (1.16) -0.29 (-0.49, -0.08) 0.006 -0.34 (-0.54, -0.13) 0.002 Weight-for-age z-score -0.55 (1.01) -0.69 (1.07) -0.14 (-0.33, 0.04) 0.135 -0.21 (-0.40, -0.02) 0.041 Weight-for-length z-score 0.10 (1.13) 0.21 (1.07) 0.10 (-0.11, 0.31) 0.330 0.05 (-0.17, 0.26) 0.663 Head circumference z-score -0.15 (1.08) -0.25 (1.20) -0.10 (-0.30, 0.10) 0.337 -0.13 (-0.34, 0.08) 0.223 a Values are means (standard deviations) unless otherwise indicated b Differences in means and P-values were estimated using linear regression models Models were adjusted for the following maternal factors: age, education, proxy score for socio-economic status (SES), body-mass-index, mid-upper arm circumference, anaemia at enrolment and Plasmodium falciparum infection at enrolment. https://doi.org/10.1371/journal.pone.0215760.t002 Table 3. Adverse pregnancy outcomes by HIV infection status a . Outcomes No HIV (N = 833) HIV (N = 134) Unadjusted Risk ratio (95% CI) b Pvalue b Adjusted Risk Ratio (95% CI) b Pvalue b Inadequate gestational weight gain c 597 (72.2) 99 (73.9) 1.02 (0.83, 1.27) 0.831 1.06 (0.85. 1.33) 0.594 Preterm birth (<37 gestation weeks) 47 (5.6) 16 (11.9) 2.12 (1.24, 3.62) 0.006 1.99 (1.14, 3.46) 0.015 Low birth weight (<2500g) 98 (11.8) 26 (19.4) 1.65 (1.11, 2.44) 0.012 1.81 (1.20, 2.73) 0.005 Newborn stunting (LAZ<-2) 124 (14.9) 33 (24.6) 1.65 (1.18, 2.32) 0.003 1.87 (1.24, 2.83) 0.003 Newborn underweight (WAZ<2) 56 (6.7) 13 (9.7) 1.44 (0.81, 2.56) 0.211 1.56 (0.86, 2.83) 0.144 Newborn wasting (WLZ<-2) 31 (3.7) 2 (1.5) 0.40 (0.10, 1.66) 0.207 0.45 (0.10, 1.93) 0.282 Newborn small head size (HCZ<-2) 31 (3.7) 9 (6.7) 1.80 (0.88, 3.71) 0.108 1.81 (0.86, 3.80) 0.119 Small-for-gestational-age (birth weight below 10 th percentile of birth weight for age (Intergrowth Standards) 235 (28.2) 55 (41.0) 1.45 (1.16, 1.83) 0.001 1.66 (1.21, 2.26) 0.001 a Values are n (%) unless otherwise indicated b Risk ratio and P-values were estimated using log binomial models or Poisson regression models c Below the lower cutoff of the Institute of Medicine’s recommended range [16] All models were adjusted for age, education, proxy score for socio-economic status (SES), body-mass-index, mid-upper arm circumference, anaemia at enrolment and Plasmodium falciparum infection at enrolment. https://doi.org/10.1371/journal.pone.0215760.t003 Lipid based nutrient supplements, maternal HIV infection and adverse pregnancy outcomes PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 6 / 15 attenuated after excluding those who were on ART whereas the association between maternal HIV and PTB strengthened. We did not collect information on the exact ARV drugs received by the individual HIV positive women participating in the study. However, in general the ARV regimen for pregnant women in Malawi at the time of the study included stavudine, lamivudine, nevirapine, zidovudine, efavirenz, tenofovir and lopinavir/ritonavir. Since private ART care was rare in the area it is likely that most of the women were on this regimen. There was one case of spina bifida identified in an infant born to an HIV positive woman. Because previous studies conducted in the pre-ART era showed that HIV was associated with a smaller head circumference [20], we explored whether the mean head circumference differed according to maternal ART use in our study. After adjusting for possible confounders, our results showed that there was no difference in the mean head circumference of infants born to HIV positive mothers who were receiving ART when compared to ART naïve HIV positive mothers (Mean difference HCZ (95% CI), 0.23 (-0.23, 0.70); P = 0.332). Since there was a considerable loss to follow up, we performed an intention to treat analysis where all individuals excluded from the analysis because of miscarriages, stillbirth or incomplete/improbable data were assumed to have had a poor outcome. Those with twin gestation or missing HIV results were not included in this analysis because twin gestation itself is a risk factor for adverse outcomes and HIV status was required as the independent variable. Out of the 1322 participants included in the sensitivity analysis, 645 infants (including 336 who were defined as being SGA) were considered to have FGR. Maternal HIV infection was associated with FGR (1.27 (1.01, 1.59, P = 0.039). Table 4. Continuous pregnancy outcomes by HIV status, stratified by intervention group a . Outcome No HIV Mean (95% CI) HIV Mean (95% CI) Adjusted difference in means (95% CI) b P-value b PFDR c Gestational weight gain rate, (g/week) LNS 298 (105) 278 (105) -20 (-53, 13) 0.231 0.231 MMN 298 (101) 263 (113) -42 (-76, -7) 0.017 0.051 IFA 297 (101) 276 (107) -29 (-62, 4) 0.082 0.123 Mean (SD) birth weight, g LNS 2996 (420) 2988 (478) -143 (-125, 153) 0.841 0.841 MMN 3004 (426) 2822 (457) -192 (-339, -45 0.011 0.033 IFA 2962 (430) 2826 (394) -151 (-289, -12) 0.033 0.051 Mean (SD) length-for-age z-score LNS -0.95 (1.04) -0.98 (0.91) -0.02 (-0.35, 0.31) 0.918 0.918 MMN -0.96 (1.08) -1.38 (1.33) -0.42 (-0.80, to -0.03) 0.035 0.053 IFA -1.05 (1.16) -1.47 (1.19) -0.52 (-0.89, -0.14) 0.007 0.021 Mean (SD) weight-for-age z-score LNS -0.51 (0.96) -0.37 (0.95) 0.10 (-0.21, 0.41) 0.516 0.516 MMN -0.53 (1.01) -0.92 (1.10)) -0.40 (-0.76, -0.05) 0.027 0.081 IFA -0.60 (1.05) -0.83 (1.08) -0.31 (-0.65, 0.023) 0. 067 0.101 LNS, lipid-based nutrient supplements; MMN, multiple micronutrient supplements; IFA, iron folic acid; and PFDR, positive false discovery rate. Women with no HIV: LNS (N = 271); MMN (N = 287) and IFA (N = 275). Women with HIV: LNS(N = 46), MMN (39), IFA (49). a Values are means (standard deviations) unless otherwise indicated b Differences in means and P-values were estimated using linear regression models c P value adjusted for multiple testing using false discovery rate method [18] All models were adjusted for age, education, proxy score for socio-economic status (SES), body-mass-index, mid-upper arm circumference, anaemia at enrolment and Plasmodium falciparum infection at enrolment https://doi.org/10.1371/journal.pone.0215760.t004 Lipid based nutrient supplements, maternal HIV infection and adverse pregnancy outcomes PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 7 / 15 Table 5. Adjusted dichotomous pregnancy outcomes by HIV status, stratified by intervention group a . Outcome No HIV N (%) HIV N (%) Risk Ratio (95% CI) b P-value b PFDR c Preterm birth (<37 gestation weeks) LNS 13 (4.8) 2 (4.4) 0.96 (0.20, 4.51) 0.958 0.958 MMN 15 (5.2) 7 (17.5) 4.79 (1.84, 12.5) 0.001 0.003 IFA 19 (6.9) 7 (14.3) 1.83 (0.68, 4.94) 0.235 0.235 Low birth weight (<2500g) LNS 31 (11.4) 8 (17.4) 1.74 (0.75, 4.03) 0.193 0.243 MMN 34 (11.9) 7 (17.5) 1.66 (0.71, 3.87) 0.243 0.243 IFA 33 (12.0) 11 (22.5) 1.94 (0.91, 4.96) 0.088 0.243 Newborn stunting (LAZ<-2) LNS 43 (15.9) 6 (13.0) 0.84 (0.34, 2.03) 0.691 0.691 MMN 38 (13.2) 11 (27.5) 2.40 (1.15, 4.98) 0.019 0.029 IFA 43 (15.6) 16 (32.7) 2.40 (1.26, 4.59) 0.008 0.024 Small-for-gestational-age (birth weight below 10th percentile of birth weight for age (Intergrowth Standards) LNS 81 (29.9) 15 (32.6) 1.13 (0.64, 2.02) 0.688 0.688 MMN 75 (26.1) 16 (40.0) 1.65 (0.91, 2.97) 0.098 0.147 IFA 79 (28.7) 25 (51.0) 2.12 (1.29, 3.47) 0.003 0.099 LNS, lipid-based nutrient supplements; MMN, multiple micronutrient supplements; IFA; iron folic acid; and PFDR, positive false discovery rate. Women with no HIV: LNS (N = 271); MMN (N = 287) and IFA (N = 275). Women with HIV: LNS(N = 46), MMN (39), IFA (49). a Values are n (%) unless otherwise indicated b Risk ratio and P-values were estimated using log binomial models or Poisson regression models c P value adjusted for multiple testing using false discovery rate method [18] All models were adjusted for age, education, proxy score for socio-economic status (SES), body-mass-index, mid-upper arm circumference, anaemia at enrolment and Plasmodium falciparum infection at enrolment https://doi.org/10.1371/journal.pone.0215760.t005 Table 6. Sensitivity analysis: HIV and continuous pregnancy outcomes after excluding HIV positive women who were on antiretroviral therapy a . Outcome No HIV (N = 833) HIV (N = 134) Unadjusted difference in means (95% CI) b P-value b Adjusted difference in means (95% CI) b P-value b Gestational weight gain rate, g/ week 297 (102) 263 (101) -34 (-56, -13) 0.002 -36 (-57, -14) 0.001 Duration of pregnancy, weeks 39.4 (1.60) 39.1 (1.76) -0.33 (-0.67, 0.01) 0.061 -0.34 (-0.69, 0.00) 0.052 Birth weight, g 2987 (425) 2890 (446) -98 (-188, -7) 0.034 -123 (-213, -32) 0.008 Length-for-age z-score -0.99 (1.10) -1.25 (1.092) -0.27 (-0.50, 0.04) 0.024 -0.33 (-0.56, -0.10) 0.005 Weight-for-age z-score -0.55 (1.01) -0.71 (1.02) -0.16 (-0.38, 0.05) 0.131 -0.27 (-0.51, -0.02) 0.036 Weight-for-length z-score 0.10 (1.13) 0.12 (1.07) 0.02 (-0.22, 0.26) 0.868 -0.01 (-0.25, 0.24) 0.957 Head circumference z-score -0.15 (1.08) -0.30 (1.11) -0.15 (-0.37, 0.08) 0.213 -0.17 (-0.41, 0.06) 0.143 a Values are means (standard deviations) unless otherwise indicated b Differences in means and P-values were estimated using linear regression models https://doi.org/10.1371/journal.pone.0215760.t006 Lipid based nutrient supplements, maternal HIV infection and adverse pregnancy outcomes PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 8 / 15 Discussion We investigated the association between maternal HIV and pregnancy outcomes, and whether this association differed across nutrient intervention groups in a cohort of women participating in a nutrient supplementation trial in southern rural Malawi. The study findings show that maternal HIV status was inversely associated with weekly GWG, birth weight, LAZ, and WAZ and positively associated with PTB, LBW, stunting and being born SGA. HIV infected women who received LNS tended to have values of birth size or risk of adverse outcomes similar to that of HIV uninfected women regardless of the type of supplement received by the uninfected women. The association between HIV infection and PTB and newborn stunting differed according to the type of dietary supplementation received during pregnancy. Maternal HIV was not associated with PTB if the mothers received LNS or IFA; or newborn stunting if the mothers received LNS. The main weakness of our study was that there was significant loss to follow up whereby only 70% of participants were eligible for inclusion in the analysis. Additionally, there were differences in some of the baseline characteristics between those who were included and those excluded in the analysis. The differences in socioeconomic status score, age and BMI were unlikely to have affected our results because socioeconomic status score was not associated with birth weight in this population and the differences in age and BMI were probably too small to be clinically meaningful. However, considering that the proportions of primigravidity and anaemia were much higher among the excluded women and that they are both associated with foetal growth faltering, we may have underestimated the impact of HIV on birth size. However, after controlling for characteristics that differed between the included and excluded participants, our adjusted results did not change for the most part. Further, the rate of attrition did not differ by HIV infection and intention-to-treat-analysis supported the findings that HIV was associated with FGR. A limitation of these analyses was that the main iLiNS-DYAD-Malawi trial was not powered for the stratified analyses and the study may have lacked sufficient power to detect differences in the incidence of relatively uncommon dichotomous outcomes such as PTB and LBW. Therefore, failure to observe a significant association between HIV and PTB or LBW among women who received IFA or MMN (for LBW only) needs to be interpreted with caution. Table 7. Sensitivity analysis: Adjusted adverse pregnancy outcomes by HIV infection status after excluding HIV positive women who were on antiretroviral therapy a . Outcomes No HIV (N = 833) HIV (N = 134) Unadjusted Risk ratio b (95% CI) Pvalue b Risk Ratio (95% CI) b Pvalue b Inadequate gestational weight gain c 597 (72.2) 74 (77.1) 1.07 (0.84, 1.36) 0.595 1.11 (0.87, 1.42) 0.399 Preterm birth (<37 gestation weeks) 47 (5.6) 1313.58) 2.40 (1.35, 4.27) 0.003 2.34 (1.30, 4.19 0.004 Low birth weight (<2500g) 98 (11.8) 17 (17.7) 1.50 (0.94, 2.41) 0.088 1.62 (1.00, 2.63) 0.049 Newborn stunting (LAZ<-2) 124 (14.9) 24 (25.0) 1.68 (1.15, 2.46) 0.008 1.93 (1.23, 3.05 0.004 Newborn underweight (WAZ<2) 56 (6.7) 8 (8.3) 1.24(0.61 2.52) 0.553 1.31 (0.63, 2.71) 0.465 Newborn wasting (WLZ<-2) 31 (3.7) 2 (2.08) 0.56 (0.14, 2.30) 0.421 0.56 (0.13, 2.40) 0.438 Newborn small head size (HCZ<-2) 31 (3.7) 5 (5.2) 1.40 (0.56, 3.51) 0.474 1.37 (0.53, 3.50) 0.514 Small-for-gestational-age (birth weight below 10 th percentile of birth weight for age (Intergrowth Standards) 235 (28.2) 39 (40.6) 1.44 (1.10, 1.88) 0.007 1.62 (1.14, 1.30) 0.007 a Values are n (%) unless otherwise indicated b Risk ratio and P-values were estimated using log binomial models or Poisson regression models https://doi.org/10.1371/journal.pone.0215760.t007 Lipid based nutrient supplements, maternal HIV infection and adverse pregnancy outcomes PLOS ONE | https://doi.org/10.1371/journal.pone.0215760 May 2, 2019 9 / 15